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 Insect Science (2010) 17, 163–165, DOI 10.1111/j.1744-7917.2010.01348.x
PREFACE
Exploring and integrating cellulolytic systems of insects
to advance biofuel technology
Jian-Zhong Sun1 and Michael E. Scharf 2
1
Biofuels Institute, School of the Environment, Jiangsu University, Zhenjiang 212013, Jiangsu, China. Email: jzsun1002@hotmail.com
or jzsun1002@ujs.edu.cn 2 Entomology and Nematology Department, PO Box 110620, Bldg 970 Natural Area Dr., University of Florida,
Gainesville, FL 32611-0620, USA. Email: mescharf@ufl.edu
In line with the requirements for sustainable economies
and clean environments, cellulose-based biofuels have
recently received tremendous attention both in industry
and academic communities worldwide. Alternative and
renewable fuels derived from lignocellulosic biomass of-
fer the potential to reduce our dependence on fossil fuels
and mitigate global climate change. The world, therefore,
is on the verge of an unprecedented increase in the pro-
duction and use of biofuels. However, in industry, break-
through technologies to overcome barriers to developing
cost-effective processes for converting biomass to fuels
and chemicals are not yet fully realized. It is worthy to
note that, over the past two decades, industrial bioethanol
technology has mainly been based on biocatalysis and
fermentation technologies from bacterial and fungal cel-
lulolytic systems, in combination with breakthroughs in
molecular genetics, enzyme engineering and metabolic
engineering. In practice, the current state of technology
with respect to biomass conversion is still far away from
being mature for large scale application due to its effi-
ciency and processing economics. To improve upon our
current technology, it seems that we need to review our
ongoing strategy and explore/learn from other sound cel-
lulolytic systems in nature, such as wood-feeding termites
or other insects. Such insects can process lignocellulosic
biomass much more efficiently with their highly special-
ized gut systems, which can truly be considered as highly
efficient natural bioreactors.
With some of the most intractable issues facing the
world regarding efficient and economic conversion of cel-
lulosic biomass, this special publication comes at a critical
and timely moment.
Most insects are unable to use lignocellulosic substrates
as their main food sources, but some insects subsist on
lignocellulosic biomass as their only foods. The types

C 2010 The Authors
Journal compilation 
C Institute of Zoology, Chinese Academy of Sciences
of biomass fed upon by cellulolytic insects range from
agricultural crops to forest woody substrates, such as in
the case of termites (all seven families), wood-feeding
roaches (Blattidae, Cryptoceridae), beetles (Anobiidae,
Buprestidae, Cerambycidae, Scarabaeidae), wood wasps
(Siricidae), leaf-shredding aquatic insects (Pteronarci-
dae, Limnephilidae, Tipulidae), silverfish (Lepismati-
dae), leaf-cutting ants (Formicidae), and so on. Cellulose
digestion has been demonstrated in more than 20 insect
families representing ten distinct insect orders, for ex-
ample, Thysanura, Plecoptera, Dictyoptera, Orthoptera,
Isoptera, Coleoptera, Trichoptera, Hymenoptera, Phas-
mida and Diptera. The ability of these insects to feed
on wood, foliage and detritus has recently stimulated ex-
tensive investigation into the mechanisms of how these
insects digest the structural and recalcitrant lignocellu-
lose in their foods, as well as their potential to advance
current biofuel technologies and processing. Recent stud-
ies using advanced molecular biotechnologies, such as
metagenomics, proteomics, transcriptomics, and so on,
have brought new insights into the mechanisms of biomass
deconstruction within these small, but complicated insect
gut systems. It has been reported that the digestion ef-
ficiency of wood-feeding termites is 74%–99% for cel-
lulose and 65%–87% for hemicellulose, which mainly
function via a collaboration between two catalyst sys-
tems: (i) termite endogenous catalyst systems, and (ii)
catalysts from a variety of gut symbiotic microorgan-
isms, including cellulolytic protozoa and bacteria. The
number of the novel cellulases and hemicellulases, as
well as the associated encoding genes from a variety of
cellulose-feeding insects has been continuously updated
in recent years. Descriptions of lignase enzymes and the
genes that code them have been lacking; however, recent
findings have suggested several viable candidates in both
163
164
areas. Screening of the genes/enzymes showing suitable
properties for industrial applications is one of the impor-
tant applied goals behind the exploration of insect cel-
lulolytic systems. Besides unveiling the mystery of the
insect catalyst systems on cellulosic substrates, studies on
physiochemical microhabitats of insect gut systems have
also shed new light on better understanding of what types
of gut environments may actually support an efficient
cellulolytic system. Clearly, all these investigations are
theoretically and practically substantial for understand-
ing insect catalyst systems and advancing current biofuel
technology. To facilitate the integration of these two dif-
ferent areas, this special issue bridges the gap between
investigations of insect cellulolytic systems and their po-
tential applications for biofuel refinery operations. This
issue, therefore, covers a range of recent experimental ad-
vancements with seven original research papers and five
review papers that address the current state of the art in
research, methodology and application, as well as various
insect cellulolytic systems.
Critical to the efficiency of biomass processing in the
biofuel industry are pretreatment technologies that are de-
veloped for a variety of the diverse feedstocks that are cur-
rently available. Pretreatment regimes must be designed
to remove substrate-specific barriers to cellulases to im-
prove cellulose digestion. A review article by Scharf and
Boucias (2010) reviews recent findings from research into
the host termite transcriptome that have revealed candi-
date enzymes (lignases and phenolic acid esterases) to
modify lignin components from lignocellulosic substrate;
and they also discuss research needs and opportunities
for consideration by entomologists working in this area.
Furthermore, Ke et al. (2010) report on oxygen profiling
in situ within the fore-, mid- and hindgut of two wood-
feeding lower termite species (characterized by the pres-
ence of symbiotic protists residing in hindgut) and also
confirm that lignin modification/disruption mainly oc-
curs within termite fore- and midgut compartments, after
which the wood particles then move to the hindgut for
further depolymerization by protozoa residing in hindgut.
This preconditioning processing on lignin components
likely permits greater access to cellulose.
With regard to developing novel cellulase biocatalysts
based on genes discovered from wood-feeding lower ter-
mites, a paper by Zhang et al. (2010) describes two re-
combinant endogenous glycosyl hydrolases from a lower
termite species, expressed in Escherichia coli that func-
tionally convert cellulose to glucose. This work will surely
help scientists optimize recombinant cellulolytic enzyme
production and combinations for biomass conversion. Cao
et al. (2010) report on examinations of hydrogen/methane
emission from three lower termite species as byprod-
Journal compilation 
C Institute of Zoology, Chinese Academy of Sciences, Insect Science, 17, 163–165
ucts produced during the course of cellulose degradation.
These findings imply a unique mechanism for producing
biohydrogen as a by-product during cellulose conversion
through gut cellulolytic and metabolic systems. To bet-
ter understand the symbiotic composition differences be-
tween gut and nest-associated microbial communities in
fungus-growing termites (a higher termite species), Long
et al. (2010) report differences in composition for both
fungal and bacterial communities between two different
symbiotic microhabitats. The paper by Long et al. (2010)
contributes to a better understanding of the potentially in-
tegrated functions played by in-gut symbionts and external
“ecto”-symbionts during the wood degradation process.
Apart from the wood-feeding termites, Geib et al.
(2010) report on enzyme biochemistry in a wood-feeding
beetle, the Asian longhorn beetle, relevant to lignocel-
lulose degradation. Geib et al. (2010) used zymogram
analysis to identify and characterize cellulases and hemi-
cellulases active against cellulose and hemicellulose sub-
strates. Because this beetle feeds on a range of tree species
and uses them as sole food sources, mining this insect
gut for lignocellulases can potentially yield new enzymes
for processing lignocellulolytic material into cellulosic
biofuels. For another cellulose-consuming beetle species,
Huang et al. (2010) review the physiochemical properties
of the scarab beetle gut (larval stage), the diversity and di-
gestive roles that symbiotic microflora play in the scarab
gut, and they further discuss the potential for applying
these digestive processes in artificial bioreactors.
Exploring another specific cellulose-consuming insect
from the order Diptera (crane fly), which is a leaf shred-
ding aquatic insect that lives in forested ecosystems,
Rogers and Doran-Peterson (2010) report on the analy-
sis of cellulolytic and hemicellulolytic enzyme activity
within this insect gut (larval stage). Rogers and Doran-
Peterson (2010) also report on identification and char-
acterization of a novel cellulolytic bacterial species iso-
lated from its gut system. In a related report from the
same laboratory, Cook and Doran-Peterson (2010) show
the potential of the crane fly gut to serve as a natural
biorefinery model to apply in improving and developing
biomass-to-biofuel technology.
The advancement of genomics and proteomics research
tools are expected to allow new insights into the mech-
anisms for wood deconstruction by cellulose-feeding in-
sects, as well as facilitate the discovery of new cellulolytic
enzymes from a wide range of cellulolytic systems. On
this topic, Willis et al. (2010) review the diverse method-
ologies used to detect, quantify, clone and express cellu-
lolytic enzymes from insects, as well as their advantages
and limitations. In addition, Shi et al. (2010) also provide
a comprehensive review on molecular approaches to study

C 2010 The Authors

insect gut symbiotic microbiota with a variety of “omics”
tools. Last, the contribution by Landis and Werling (2010)
addresses pest management and landscape ecology issues
in relation to biofuel crop production; specifically, po-
tential arthropod community responses to a large scale
production of biofuel crops and biomass harvesting from
North American forests. In this review paper, some crit-
ical topics relevant to the arthropod ecology in impacted
systems are also proposed to meet the coming challenges
associated with a large-scale planting of energy crops and
forest biomass harvesting. This is an important emerg-
ing area of research stemming from renewed interests in
biofuels that is creating opportunities for entomologists.
In summary, this special issue focuses on broad-ranging
areas of progress and challenges associated with the uti-
lization of gene, catalyst, and other unique mechanisms
from wood-feeding insects, as well as ecological impacts
and pest management needs of biofuel-based agriculture
and forestry. This special issue comes at a time when
government and industry are scaling up their investment
in biofuels, with the expectation of a long-term need for
alternatives to petroleum-based liquid fuels. Exploring
insect cellulolytic systems will lead to the discovery of
a variety of novel biocatalysts and genes that encode
them, as well as associated unique mechanisms for ef-
ficient biomass conversion. This new and evolving multi-
disciplinary area has emerged between insect and bioengi-
neering sciences; without question, it will pave the way for
future breakthroughs and innovations in associated areas
of industrial biotechnology. It is also hoped that this spe-
cial issue can promote productive collaborations between
scientists working in the various disciplines represented
herein.
We hope that readers will find these articles interesting
and helpful to their research efforts. It has been our plea-
sure to put together this special issue in Insect Science. We
would like to thank Professor Le Kang, Editor-in-Chief,
for providing researchers a unique forum in which to re-
port ongoing research activities on insect cellulolytic sys-
tems and their potential to update the current technology
for biomass processing. We would also like to sincerely
thank Dr. Yun Xian Zhao for expert editorial assistance,
and all of the authors that have contributed to this special
issue for their dedicated efforts and excellent contribu-
tions. We acknowledge with appreciation the assistance
of the reviewers who have given their valuable support
and expertise in reviewing manuscripts submitted for this
special issue. The quality of the issue can be attributed in
large measure to the quality of their efforts, for which we
are sincerely grateful.

C 2010 The Authors
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C Institute of Zoology, Chinese Academy of Sciences, Insect Science, 17, 163–165
165
References
Cao, Y., Sun, J.Z., Rodriguez, J.M. and Lee, K.C. (2010) Hy-
drogen emission by three wood-feeding subterranean termite
species (Isoptera: Rhinotermitidae): Production and charac-
teristics. Insect Science, 17, 237–244.
Cook, D.M. and Doran-Peterson, J. (2010) Mining diversity of
the natural biorefinery housed within Tipula abdominalis lar-
vae for use in an industrial biorefinery for production of lig-
nocellulosic ethanol. Insect Science, 17, 303–312.
Geib, S.M., Tien, M. and Hoover, K. (2010) Identification of
proteins involved in lignocellulose degradation using in gel
zymogram analysis combined with mass spectroscopy-based
peptide analysis of gut proteins from larval Asian longhorned
beetles, Anoplophora glabripennis. Insect Science, 17, 253–
264.
Huang, S.W., Zhang, H.Y., Marshall, S. and Jackson, T.A. (2010)
The scarab gut: A potential bioreactor for bio-fuel production.
Insect Science, 17, 175–183.
Ke, J., Sun, J.Z., Nguyen, H.D., Singh, D., Lee, K.C., Beyenal,
H. and Chen S.L. (2010) In-situ oxygen profiling and lignin
modification in guts of wood-feeding termites. Insect Science,
17, 277–290.
Landis, D.A. and Werling, B.P. (2010) Arthropods and biofuel
production systems in North America. Insect Science, 17,
220–236.
Long, Y., Xie, L., Liu, N., Yan, X., Li, M.H., Fan, M.Z. and
Wang, Q. (2010) Comparison of gut-associated and nest-
associated microbial communities of a fungus-growing ter-
mite (Odontotermes yunnanensis). Insect Science, 17, 265–
276.
Rogers, T.E. and Doran-Peterson, J. (2010) Analysis of cellu-
lolytic and hemicellulolytic enzyme activity within the Tipula
abdominalis (Diptera: Tipulidae) larval gut and characteri-
zation of Crocebacterium ilecola gen. nov. sp. nov., isolated
from the Tipula abdominalis larval hindgut. Insect Science,
17, 291–302.
Scharf, M.E. and Boucias, D.G. (2010) Potential of termite-
based biomass pre-treatment strategies for use in bioethanol
production. Insect Science, 17, 166–174.
Shi, W.B., Syrenne, R., Sun, J.Z. and Yuan, J.S. (2010) Molecular
approaches to study the insect gut symbiotic microbiota at the
‘omics’ age. Insect Science, 17, 199–219.
Willis, J.D., Oppert, C. and Jurat-Fuentes, J.L. (2010) Methods
for discovery and characterization of cellulolytic enzymes
from insects. Insect Science, 17, 184–198.
Zhang, D., Lax, A.R., Bland, J.M., Yu, J., Fedorova, N. and
Nierman, W.C. (2010) Hydrolysis of filter-paper cellulose to
glucose by two recombinant endogenous glycosyl hydrolases
of Coptotermes formosanus. Insect Science, 17, 245–252.
 Insect Science (2010) 17, 166–174, DOI 10.1111/j.1744-7917.2009.01309.x

REVIEW

Potential of termite-based biomass pre-treatment strategies

for use in bioethanol production

Michael E. Scharf and Drion G. Boucias

Entomology and Nematology Department, University of Florida, Gainesville, Florida, USA

Abstract When considering the current state of the biorefinery industry, it is read-
ily apparent that industrial cellulose and hemicellulose digestion processes are relatively
advanced, whereas enzymatic pre-treatment strategies for biomass delignification and cel-
lulose solubilization are not well developed. The need for efficient biomass pre-treatment
strategies presents a significant opportunity for researchers studying lignocellulose diges-
tion in termites and other insects. With an emphasis on industrial biomass pre-treatment,
this review provides an overview of: (i) industrial biorefining operations (feedstocks, pro-
cessing, and economics); (ii) recent findings from termite research that have revealed
candidate enzymes; and (iii) research needs and opportunities for consideration by en-
tomologists working in this area. With respect to research findings, recently identified
candidate lignases (laccases, catalases, peroxidases, esterases), other potentially important
detoxification enzymes (cytochrome P450, superoxide dismutase), and phenolic acid es-
terases (carboxylesterases) that may assist in hemicellulose solubilization are overviewed.
Regarding research needs and opportunities, several approaches for identification of can-
didate pre-treatment enzymes from upstream, symbiont-free gut regions are also described.
Key words bioenergy, bioethanol, biorefining, Isoptera, lignocellulose, renewable
energy

Termites, lignocellulose, and review objectives
Termites are unique animals in that they have become
specially adapted to survive on sugars and other nu-
trients obtained from otherwise nutritionally poor lig-
nocellulose diets (Ohkuma, 2003). Lignocellulose is
a mixture of the three plant-produced polymers: cel-
lulose, hemicellulose and lignin (structures provided
in Scharf & Tartar, 2008). Cellulose consists of ex-
tended β-1,4-linked glucose polymers that are held
together in bundles by hemicellulose (Ljungdahl &
Erickson, 1985; Lange, 2007). Hemicellulose is com-
posed of more compact β-1,4-linked polymers of 5- and
Correspondence: Michael E. Scharf, Entomology and Ne-
matology Department, PO Box 110620, University of Florida,
Gainesville, Florida 32611-0620, USA. Tel: 352 273 3954; fax:
352 392 0190; email: mescharf@ufl.edu
6-carbon sugars and their uronic acids (Saha, 2003).
Lignin is a 3-dimensional polymer of phenolic compounds
(coumaryl, coniferyl and sinapyl alcohol) that are linked
to each other and to hemicellulose by ester bonds. An-
other attribute of hemicellulose is its esterification with
monomers and dimers of phenolic acid esters that are
synonymous with the mono-lignols that compose lignin
(Saha, 2003; Crepin et al., 2004; Benoit et al., 2008).
Hemicellulose is much more water-soluble than cellulose,
a property that has been exploited in industrial biorefinery
operations (see later).
Termites digest lignocellulose by using endogenous and
symbiont-produced digestive enzymes (Breznak & Brune,
1994; Watanabe et al., 1998; Ohkuma, 2003; Scharf &
Tartar, 2008; Tartar et al., 2009). Termite gut symbionts
consist of diverse micro-organisms such as protozoa,
bacteria, spirochetes, fungi and yeast (Cleveland, 1923;
Breznak & Brune, 1994; Ohkuma, 2003; Brune, 2006;
Warnecke et al., 2007). The order Isoptera is divided into


C 2010 The Authors 166
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C Institute of Zoology, Chinese Academy of Sciences

the higher and lower termites based mostly on symbiont
composition. Lower termites, including Reticulitermes
flavipes, are hosts to both cellulolytic protozoa and a va-
riety of non-cellulolytic prokaryotes (Lewis & Forschler,
2004; Fisher et al., 2007). Higher termites, alternatively,
lack protozoa but serve as hosts to a diverse pool of cel-
lulolytic and non-cellulolytic prokaryotes (Wood & John-
son, 1986; Warnecke et al., 2007).
In recent years, significant research efforts have been
directed at understanding termite lignocellulose digestion.
The primary reason for this major research thrust is the
need for renewable bioenergy. This review addresses one
component of renewable bioenergy production: biomass
pre-treatment. The objectives of this review are to: (i) pro-
vide an overview of industrial lignocellulose processing
with respect to feedstocks, processing, and economics;
(ii) review recent findings from termite research that have
revealed candidate biomass pre-treatment enzymes; and
(iii) summarize pre-treatment research needs and opportu-
nities for consideration by scientists working in the areas
of insect nutrition and lignocellulose digestion.
Industrial biorefining: lignocellulose
feedstocks, processing, and economics
Overview
The major processes involved in industrial bioethanol
production from lignocellulosic feedstocks are shown
in Figure 1. Lignocellulose can serve as a precursor
for a number of biorefinery processes such as cellu-
lose/ hemicellulose hydrolysis, pyrolysis, and gasification
(Ragauskas et al., 2006; Brown, 2006; Lange, 2007; Yang
& Wyman, 2008). However, all three processes depend
on biomass pre-treatment strategies that use chemicals
and/or heat to delignify feedstocks or to release the sugar
polymers cellulose and hemicellulose (Galbe & Zacchi,
FEEDSTOCK HEMICELLULOSE
BIOMASS HYDROLYSIS
PROCESSED
PRE- SUGAR
BIOMASS
TREATMENT FERMENTATION
CELLULOSE
HYDROLYSIS
Fig. 1 Flow diagram showing the major processes involved in bioethanol production from second generation (non-food) feedstocks.
Black boxes with white text indicate biorefinery processes that are the focus of the current article, and which account for ∼40%–45%
of total processing costs. Dashed lines indicate pathways for value-added by-products, energy generation, and waste recycling/disposal.
Modeled after Wyman (2001), Ragauskas et al. (2006), Galbe et al. (2007) and Yang & Wyman (2008).

C 2010 The Authors
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C Institute of Zoology, Chinese Academy of Sciences, Insect Science, 17, 166–174
Termite-based biomass pre-treatment strategies 167
2007). Current pre-treatment strategies are both chemical-
and energy-intensive, and they are the most costly com-
ponent of bioethanol production (Wooley et al., 1999a,
1999b; Aden et al., 2002; Brown, 2006; Galbe et al.,
2007; Yang & Wyman, 2008). After pre-treatment, sim-
ple sugar release is accomplished via enzymatic hydroly-
sis of β-glycosidic bonds in cellulose and hemicellulose.
The release of these fermentable simple sugars is another
highly costly phase of bioethanol production (Galbe et al.,
2007; Yang & Wyman, 2008). Energy and cost inputs
into pre-treatments and hydrolysis can be greatly reduced
with effective use of recombinant enzyme technology
(Ragauskas et al., 2006; Yang & Wyman, 2008). In partic-
ular, enzymes from lignocellulose-digesting insects and
their symbionts, especially termites, are useful for pre-
treatment and for downstream carbohydrate processing
(Rubin, 2008; Scharf & Tartar, 2008).
Lignocellulose feedstocks
At present, bioethanol production relies mostly on
food-grade sugars and starches obtained from agricul-
tural crops, for example, sugar cane (sucrose) and maize
(starch). However, if bioethanol is to compete in the
marketplace with petroleum fuels, second-generation
feedstocks will have to be developed. Second gener-
ation feedstocks are those not derived from grain or
sugar-based agricultural commodities (Wyman, 2001;
Simmons et al., 2008). There are many second-generation
feedstocks emerging from both forestry and agricul-
tural sectors, all of which are viable candidates for
consideration and development (Simmons et al., 2008).
Combined second-generation feedstock quantities po-
tentially available for bioethanol processing are esti-
mated at 1.3 billion tons per year in the US (0.3
forestry + 1.0 agricultural; Perlack et al., 2005).
Emerging forestry feedstocks include slash/loblolly pine,
LIQUID WASTE (1) DISCHARGE,
TREATMENT (2) RECYCLE
(1) VALUE-ADDED
BY-PRODUCTS,
ETHANOL
(2) HEAT / ENERGY
RECOVERY
GENERATION
SOLID WASTE
TREATMENT
168 M. E. Scharf & D. G. Boucias

poplar/hybrid poplar, willow, silver maple, black lo- distillation. The remaining solid materials after distilla-
cust, sycamore, sweetgum and eucalyptus (Nowak et al., tion (lignin, enzymes, organisms, etc.) are used to feed
2008; Simmons et al., 2008). Agricultural feedstocks boilers, produce electricity, and/or create value-added by-
of emerging importance include corn stover (Aden, products. The resulting waste water is processed and ei-
2008), sugarcane bagasse, switchgrass, reed canary ther re-used or discharged. Remaining sludge after waste
grass, miscanthus, sorghum, and other tropical grasses water treatment feeds the boiler, and then boiler ash is
(Simmons et al., 2008). Each of these feedstocks is eco- landfilled.
nomically and strategically viable in specific regions of
the US (Nowak et al., 2008; Simmons et al., 2008). Be-
Economics of lignocellulose processing
cause of the diverse lignocellulose compositions of these
different feedstocks, pre-treatment strategies will likely
The primary challenge for bioethanol competitiveness
need to be tailored to each of the individual feedstocks
is to reduce the cost of biomass processing for con-
(Tartar et al., 2009).
verting raw feedstock materials into product. An esti-
mate from 2001 is that bioethanol could compete with
Lignocellulose processing
gasoline derived from raw petroleum costing $25/bar-
rel (Wyman, 2001). More recent estimates of this
The major steps involved in industrial bioethanol pro-
price point are not available; however, recent cost es-
duction from second-generation feedstocks are outlined in
timates for bioethanol production range from 0.13 to
Figure 1 (Wyman, 2001; Yang & Wyman, 2008). Biomass
0.81 US$/L, depending on feedstock (Galbe et al., 2007).
feedstock materials are first milled to reduce particle size
For any feedstock, approximately 40% of these total
for subsequent steps (Galbe et al., 2007; Yang & Wyman,
processing costs are linked to the three phases of pre-
2008). Essentially, this processing step is analogous to
treatment, enzyme production and enzymatic hydrolysis
the mechanical degradation that would occur via the ac-
(Wooley et al., 1999a, 1999b; Aden et al., 2002; Yang &
tion of insect/termite mandibles and the gut proventricu-
Wyman, 2008). These three phases account for approxi-
lus, in which particle sizes of <50 μ are achieved (e.g.,
mately 20%, 10%, and 10%, respectively, of total costs.
Itakura et al., 1995). Next, in the process known as pre-
Unfortunately, although cost calculation tools and
treatment, the processed feedstocks are treated to dis-
models have been developed for some feedstocks
rupt lignin and to make hemicellulose/cellulose available
(Wooley et al., 1999a; McAloon et al., 2000; Aden, 2008),
for biologic degradation. Pre-treatment can take several
bioethanol costs are dependent on many regional or lo-
forms, including biological, chemical, physical and ther-
cal factors, and thus are site-specific and geographically
mal (Yang & Wyman, 2008 and references therein). After
variable (Wyman, 2001; Galbe et al., 2007). For these
pre-treatment, hydrolysates usually contain hemicellulose
reasons, it is difficult to predict how specific advances
and solids contain cellulose. Hydrolysates are neutralized
resulting from novel insect or symbiont enzymes could
and conditioned to remove any lignin metabolites, value-
specifically impact processing economics. In particular,
added by-products, and other materials deleterious to the
of all the options proposed for enzymatic pre-treatments
fermentation process.
for lignin depolymerization (biological, chemical, physi-
Next, hemicellulose and/or cellulose are depolymerized
cal, thermal), it has long been recognized that biological
into their 5- and 6-carbon monomer sugars, respectively,
delignification would be the least energy-intensive and
by enzymes secreted from engineered fungi. In the case
most economical (Kirk & Harkin, 1973; Fan et al., 1982;
of the fungus Trichoderma, small amounts of cellulose
Detroy et al., 1980; Datta, 1981; Galbe & Zacchi, 2007;
solids or hemicellulose hydrolysates are pre-incubated
Yang & Wyman, 2008). Thus, without question, the de-
with fungal isolates to induce production of cellulases
velopment of novel enzymes clearly has the potential to
(endoglucanases and β-glucosidases) and hemicellulases,
reduce costs of pre-treatment, hydrolysis, fermentation
which are then added back to bulk pre-treated materials
and by-product enhancement/recovery.
to depolymerize cellulose into glucose. Engineered bac-
teria are used primarily for hemicellulose depolymeriza-
tion. It is also noteworthy that recombinant/engineered Candidate pre-treatment enzymes from
bacteria are also being developed for hemicellulose and R. flavipes
cellulose depolymerization purposes (Jarboe et al., 2007).
The simple sugars liberated from cellulose and hemicel- Depending on the plant species from which it is de-
lulose are fermented by many types of organisms (e.g., rived, wood lignocellulose is composed of approximately
yeast) to produce ethanol, which is then recovered by 40% cellulose, 25% hemicellulose, 20% lignin, and


C 2010 The Authors
Journal compilation 
C Institute of Zoology, Chinese Academy of Sciences, Insect Science, 17, 166–174

Laccase
Catalase
Superoxide dismutase
Glutathione peroxidase
Carboxylesterase
Cytochrome P450
0 2 4 6 8 10 12 14 16
Number of genes
Fig. 2 Summary of genes encoding candidate biomass pre-
treatment enzymes identified from a Reticulitermes flavipes gut
tissue library (Scharf & Tartar, 2008; Tartar et al., 2009). No ho-
mologous or functionally similar genes were identified from a
hindgut symbiont library. Black and gray bars, respectively, rep-
resent candidate lignases and candidate phenolic acid esterases.
15% other miscellaneous components (Ragauskas et al.,
2006; Lange, 2007 and references therein). A recent
survey of host and symbiont transcriptomes from the
R. flavipes gut revealed over 175 genes encoding cellu-
lases, hemicellulases, candidate lignases, and other poten-
tially relevant digestive enzymes (Scharf & Tartar, 2008;
Tartar et al., 2009). The following section reviews re-
search findings on two candidate groups of R. flavipes
gut enzymes with potential relevance in industrial lig-
nocellulose pre-treatment: (i) candidate lignases; and (ii)
candidate phenolic acid esterases (Fig. 2).
Candidate lignases: laccases, catalases, peroxidases,
cytochrome P450s and superoxide dismutases
The rationale for investigation of lignase activities in
termites comes from several decades of research doc-
umenting lignin modification and/or degradation ca-
pabilities in termite guts (Esenther & Kirk, 1974;
Butler & Buckerfield, 1979; Cookson, 1987, 1988;
Breznak & Brune, 1994; Brune et al., 1995a; Itakura
et al., 1995; Kuhnigk & König, 1997; Taprab et al., 2005;
Katsumata et al., 2007; Geib et al., 2008). Lignin break-
down also requires oxygen (Breznak & Brune, 1994),
which is supported by evidence that termite guts are
not completely anaerobic environments, especially the
foregut and hindgut periphery (Brune et al., 1995b). Al-
though no termite genes or gene products have yet been
proven to participate in lignin degradation, several candi-
date genes were recently identified from the R. flavipes
host gut transcriptome (Fig. 2).
Of the candidate lignases, the most relevant are lac-
cases, catalases and peroxidases, each of which is known

C 2010 The Authors
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C Institute of Zoology, Chinese Academy of Sciences, Insect Science, 17, 166–174
Termite-based biomass pre-treatment strategies 169
to degrade lignin and related phenolic compounds in fungi
(Erikkson et al., 1990; Ohkuma, 2003; Baldrian, 2006;
Anderson & Akin, 2008; Sutay-Kocabas et al., 2008). Of
these, the laccase enzyme isoform Rflac6 identified from
R. flavipes is particularly interesting. Rflac6 possesses a
secretory signal peptide, is highly expressed in salivary
gland tissue, and readily oxidizes numerous lignin-like
phenolic substrates, including the lignin monomer sinap-
inic acid (Tartar et al., 2009, unpublished results). Un-
like other insect laccases that act predominantly in cuti-
cle melanization, Rflac6 is virtually inactive toward the
melanin precursors L-DOPA and L-tyrosine (unpublished
results).
Another potentially important set of genes iden-
tified from the R. flavipes gut transcriptome en-
code a number of cytochrome P450 mono-oxygenases
(Tartar et al., 2009). The P450s are a well-represented
family of oxidative enzymes in all life-forms that are
known for their roles in detoxification and xenobi-
otic metabolism (Feyereisen, 2005). With respect to
lignin degradation, bacterial P450s have been shown
to directly oxidize lignin-like phenolic compounds
(e.g., Sutherland, 1986). Additionally, insect P450s are
well known for catalyzing O-demethylations such as
the lignin side-chain oxidations noted previously by
Geib et al. (2008) in the guts of termites and xy-
lophagous beetle larvae. Finally, because lignin diges-
tion generates potentially cytotoxic free radicals (Breznak
& Brune, 1994), antioxidant enzymes that reduce free
radicals also may have uses in industrial lignocellulose
pre-treatment. In this regard, previous sequencing work
revealed one catalase and three superoxide dismutase
(SOD)-coding genes from the R. flavipes host gut tran-
scriptome (Tartar et al., 2009). Under native conditions
in the termite gut, antioxidant enzymes such as catalases
and SODs may protect termite gut symbionts from toxic
lignin degradation products. Thus, antioxidant enzymes
may protect cellulolytic and fermentative microbes from
oxidative damage caused by lignin degradation products.
Candidate phenolic acid esterases: carboxylesterases
A number of microbial and fungal esterases recently
have been identified that enable hemicellulose solubiliza-
tion (and subsequent depolymerization) by functioning
as phenolic acid esterases (Crepin et al., 2004; Benoit
et al., 2008). These enzymes, also known as “feruloyl”
or “ferulic acid” esterases, hydrolyze carboxylester bonds
between hemicellulose sugars and lignin monomers (de
Vries et al., 1997, 2002) and possibly ester linkages in the
lignin polymer itself (Scharf & Tartar, 2008). In the case
170 M. E. Scharf & D. G. Boucias
of hemicellulose, this hydrolysis reaction constitutes a
one-step hemicellulose solubilization reaction, which has
tremendous relevance to industrial biomass pre-treatment.
Previous gut transcriptome sequencing work revealed 12
gut carboxylesterase genes from R. flavipes (Fig. 2; Tartar
et al., 2009).
The rationale for investigating termite gut car-
boxylesterases as potential phenolic acid esterases is that:
(i) the termite diet contains significant amounts of hemi-
cellulose; (ii) there are at least 45 hemicellulase genes
encoded in the R. flavipes gut meta-transcriptome; and
(iii) there is strong esterase activity and substantial iso-
form diversity in the R. flavipes gut (Tartar et al., 2009;
Wheeler et al., 2009). Four of the twelve identified es-
terase genes (named RfEst1, 2, 3 and 4) have been stud-
ied in detail (Wheeler et al., 2009). The four genes en-
code two larger juvenile hormone-like esterases (RfEst1
and 2) and two shorter esterases with homology to insect
carboxylesterases and the fungal phenolic acid esterases
(RfEst3 and 4). Gene expression studies for the four es-
terases revealed significant expression of each gene in
symbiont-free midgut, foregut, and salivary gland tissues,
with reduced hindgut expression. These findings, in ad-
dition to the fact that the esterases were only identified
from a symbiont-free gut library (not a separate sym-
biont library), and that considerable esterase activity ex-
ists in symbiont-free gut regions, strongly suggest that the
R. flavipes gut esterases are not symbiont-derived. Thus,
searches for culturable symbionts that possess phenolic
acid esterase activity, or larger-scale symbiont metatran-
scriptome sequencing are not likely to be fruitful for phe-
nolic acid esterase identification.
Research needs and opportunities
As highlighted throughout this article, the most critical
research needs in the biomass pre-treatment area cen-
ter on the development of low-cost enzymatic strate-
gies for delignification and cellulose/hemicellulose sol-
ubilization. Because pre-treatment represents the most
expensive component of bioethanol production (∼20%
of total costs), this is the area in which significant op-
portunities for innovation exist. Specifically, biologically-
based strategies are needed to replace harsh chemical and
energy-intensive heat treatments for delignification and
hemicellulose solubilization.
One area of opportunity is the prospecting of upstream
termite gut regions where biological pre-treatment natu-
rally occurs (salivary gland, foregut and midgut; Fig. 3).
It is well established that termite lignocellulose diges-
tion generally results from collaboration between host-
Journal compilation 
C Institute of Zoology, Chinese Academy of Sciences, Insect Science, 17, 166–174
E
SG
HG R
FG
MG
MT
Fig. 3 Line drawing of the Reticulitermes flavipes gut. E,
esophagus; FG, foregut; SG, salivary gland; MG, midgut; MT,
Malpighian tubules; HG, hindgut paunch; R, rectum.
and symbiont-derived digestive enzymes (reviewed by
Scharf & Tartar, 2008). For instance, the candidate host-
derived pre-treatment factors studied to date (laccases
and esterases) are produced in symbiont-free tissues, well
upstream of the symbiont-rich hindgut paunch (Fig. 3)
(Wheeler et al., 2009; Tartar et al., 2009). Thus, symbiont-
free tissues such as the salivary glands, foregut and
midgut, likely hold the greatest potential for identification
of candidate pre-treatment enzymes, model processes and
strategies.
As noted previously, no single dominant bioethanol
feedstock has yet emerged nationwide, but rather, many
regionally important feedstocks have materialized and
each is likely to remain at least regionally important
(Perlack et al., 2005; Aden, 2008; Nowak et al., 2008;
Simmons et al., 2008). Furthermore, lignocellulose com-
position varies significantly between agricultural and
forestry feedstocks, as well as within each of these two
groups (www.wisbiorefine.org; Karp & Shield, 2008; Xu
et al., 2009). Thus, another area of substantial opportu-
nity is the development of biological pre-treatment strate-
gies that are specifically tailored to regionally important
feedstocks. Selective feeding studies with live termites
would provide an excellent model system for identify-
ing feedstock-specific enzyme blends (e.g., Smith et al.,
2009). In such studies, termite colonies are fed exclusive
diets of different feedstock materials for defined time peri-
ods before gene expression and/or relevant enzyme activi-
ties are measured. Gene expression can be investigated us-
ing semi-quantitative or quantitative real-time polymerase
chain reaction (PCR) for smaller groups of genes (e.g.,
Wheeler et al., 2007), whereas broader-scale gene expres-
sion can be investigated with whole genome or gut meta-
transcriptome micro-arrays (e.g., Whitfield et al., 2002)
or next-generation sequencing (e.g., Warnecke et al.,

C 2010 The Authors

2007). Also, if desired, changes in symbiont species com-
position can be investigated using PCR-based 16S se-
quence surveys (e.g., Fisher et al., 2007; Miyata et al.,
2007; Hu et al., 2009). Finally, activities of responsive
enzymes can be investigated using model substrates that
target key digestive enzymes, for example, phenolox-
idases, esterases, hemicellulases and cellulases (Smith
et al., 2009; Tartar et al., 2009; Wheeler et al., 2007,
2009).
Finally, for optimal release of fermentable sug-
ars from specific lignocellulose feedstocks, we hy-
pothesize that co-evolved pre-treatment and cellu-
lase/hemicellulase enzymes from the same termite species
will be more efficient than mixtures of enzymes from
different organisms. For example, suites of enzymes
from a single termite digestome (Scharf & Tartar,
2008) are likely to be more efficient than mixtures of
termite pre-treatment enzymes and fungal cellulases and
hemicellulases (Tartar et al., 2009). Thus, selective feed-
ing studies that consider global digestome expression
changes and enzyme activity shifts in response to feed-
stock diets are likely to identify optimal enzyme cocktails
that work with maximal efficiency to depolymerize lig-
nocellulose feedstocks into fermentable simple sugars.
Conclusions
At ∼20% of total costs, feedstock pre-treatment is
presently the most costly component of industrial
bioethanol production. There are currently several region-
ally important bioethanol feedstocks, all in need of inno-
vations that reduce pre-treatment costs and improve pro-
cessing efficiency. This paper has provided an overview
of industrial biorefinery operations, examples of candi-
date termite-derived pre-treatment enzymes, and a scien-
tific, hypothesis-driven approach to develop novel bio-
catalyst + feedstock combinations for use in bioenergy
production.
This is a unique time for bioenergy technology devel-
opment. The selective feeding approach proposed here
can create unprecedented opportunities to develop novel
recombinant enzymes that collaborate to enable effi-
cient digestion and fermentation of bioethanol feedstocks.
Gaining such knowledge is important because it will en-
hance the development of sustainable, economically vi-
able and environmentally friendly bioethanol feedstocks.
Ultimately, developing optimal feedstock-specific biore-
finery processes can lead to significantly reduced produc-
tion costs, greater competitiveness for bioethanol in the
marketplace, and most importantly, reduced dependence
on petroleum-based energy.

C 2010 The Authors
Journal compilation 
C Institute of Zoology, Chinese Academy of Sciences, Insect Science, 17, 166–174
Termite-based biomass pre-treatment strategies 171
Acknowledgments
Research described herein was supported by a Univer-
sity of Florida – IFAS Innovation Grant to MES and
DGB, by CSREES-USDA-NRI grant No. 2007-35607-
17777 to MES, by a Phase I DOE-SBIR grant No. FG02-
08ER85063 to Chesapeake-PERL Inc.(Savage, MD, US),
and through The Consortium for Plant Biotechnology Re-
search, Inc. by DOE Prime Agreement No. DE-FG36-
02GO12026 to MES and DGB (this support does not
constitute an endorsement by DOE or by the Consortium
for Plant Biotechnology Research, Inc. of the views ex-
pressed in this publication).
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search progress in bioethanol technology. Applied Biochem-
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174 M. E. Scharf & D. G. Boucias

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Accepted November 23, 2009


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 Insect Science (2010) 17, 175–183, DOI 10.1111/j.1744-7917.2010.01320.x

REVIEW

The scarab gut: A potential bioreactor for bio-fuel production

Sheng-Wei Huang1 , Hong-Yu Zhang1 , Sean Marshall2 and Trevor A. Jackson2

1
State Key Laboratory of Agricultural Microbiology, Institute of Urban and Horticultural Pests, and Hubei Insect Resources Utilization
and Sustainable Pest Management Key Laboratory, College of Plant Science and Technology, Huazhong Agricultural University, Wuhan,
China, 2 Biocontrol, Biosecurity and Bioprocessing, AgResearch Limited, Christchurch, New Zealand

Abstract Cellulose and hemicelluloses are the most prevalent sources of carbon in
nature. Currently many approaches employ micro-organisms and their enzyme products to
degrade plant feedstocks for production of bioenergy. Scarab larvae are one such model.
They consume celluloses from a variety of sources including plant roots, soil organic matter
and decaying wood, and are able to extract nutrients and energy from these sources. In this
paper, we review the physicochemical properties of the scarab larval gut, the diversity and
digestive role that microflora play in the scarab gut and discuss the potential for applying
these digestive processes in bioreactors for improving bio-fuel production. Scarab larvae
are characterised by their highly alkaline midgut which is dominated by serine proteinase
enzymes, and a modified hindgut which harbors the majority of the intestinal microbiota
under anaerobic conditions. Evidence suggests that digestion of recalcitrant organic matter
in scarab larvae likely results from a combination of endogenous gut proteinases and
cellulolytic enzymes produced by symbiotic micro-organisms. Most of the easily digestible
proteins are mobilized and absorbed in the midgut by endogenous proteinases. The hindgut
contents of scarab larvae are characterized by high concentrations of volatile fatty acids, the
presence of fermenting bacteria, and typical anaerobic activities, such as methanogenesis.
The hindgut typically contains a wide diversity of micro-organisms, some of which appear
to be obligate symbionts with cellulolytic potential. As a result, the scarab larval gut
can be regarded as a small bioreactor resembling the rumen of sheep or cattle, where
solid food particles composed of cellulose, hemicellulose, pectin and polysaccharides
are degraded through enzymatic and fermentation processes. Together these observations
suggest scarab larvae have potential to assist the bio-fuel industry by providing new sources
of (hemi)cellulolytic bacteria and bacterial (hemi)cellulolytic enzymes.
Key words bio-fuel, bioreactor, cellulolytic enzymes, microflora, Scarabaeidae

Introduction predominate in grassland soils (Lavelle et al., 1997) and

The order Coleoptera is the largest within the insect world
and contains the family Scarabaeidae, whose members
Correspondence: Hong-Yu Zhang, State Key Laboratory of
Agricultural Microbiology, Institute of Urban and Horticul-
tural Pests, and Hubei Insect Resources Utilization and Sus-
tainable Pest Management Key Laboratory, College of Plant
Science and Technology, Huazhong Agricultural University,
Wuhan, China. Tel: +86 27 87280276; fax: +86 27 87384670;
email: zhygood@hotmail.com
other environments where decaying plant materials form
a high proportion of biomass.
The larvae of scarabaeids are almost exclusively her-
bivorous or saprophagous (Crowson, 1981), and many
species live in soils and feed on plant roots or organic
matter of low nutritive value (McQuillan & Webb, 1994;
Zhang & Jackson, 2008). The typical intestinal tract of hu-
mivorous scarabaeid beetle larvae has several characteris-
tic physicochemical properties, including: a long midgut
occupying most of the length of the body cavity (Terra,
1990); a modified and expanded hindgut (often referred


C 2010 The Authors 175
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C Institute of Zoology, Chinese Academy of Sciences
176 S. W. Huang et al.
to as a fermentation chamber) (Terra, 1990); a highly al-
kaline midgut with multiple alkali-stable hydrolases (Li
& Brune, 2005); and specific gut microbiota (Egert et al.,
2003; Zhang & Jackson, 2008). While several scarab lar-
vae are important agriculture and forestry insect pests,
many scarab larvae play an important role in decomposing
cattle dung, or decaying wood, and therefore may provide
an effective resource for allowing the utilization of other
organic biowastes in the future (Koyama et al., 2003).
In this review, we have assembled available informa-
tion on digestive enzymes and gut bacteria in scarab
larvae in order to understand the digestion process of
(hemi)celluloses (refers to both hemicelluloses and cellu-
loses) in the scarab larval gut. Through this work we hope
to offer new ideas and insights toward creating future
bioreactors for bio-fuel production.
Morphology and biochemistry of the scarab gut
Gut morphology
A typical larval alimentary tract of a Scarabaeidae is
divided into three major sections: a foregut containing a
small crop, a long midgut that occupies a high propor-
Fig. 1 View of the alimentary tract of scarab larvae showing relative locations of the foregut, midgut and hindgut for (A) Papuana
huebneri, (B) Holotrichia parallela, (C) Costelytra zealandica.
Journal compilation 
C Institute of Zoology, Chinese Academy of Sciences, Insect Science, 17, 175–183
tion of the body cavity, and a modified expanded hindgut
(Terra, 1990; Zhang & Jackson, 2008) (Fig. 1).
The foregut is lined with cuticle and extends from the
oral cavity to the cardiac valve (valvula cardiaca), which
marks the transition to the midgut (mesenteron). The crop
is expandable and can be used for food storage with cir-
cular muscles at the cardiac valve that are able to restrict
entry of food particles into the midgut. The midgut con-
sists of a tube of epithelial cells and extends for most of
the body length, occupying a large proportion of the body
cavity, and it is distinguished by the presence of three
rows of caecae circling the tract. Two rows are found at
about one-third of the distance from the anterior end and
one row is located at the terminal portion of the midgut.
The point of division between midgut and hindgut (proc-
todeum, ileum) is marked by the entry of the malphigian
tubules. The anterior portion of the hindgut is the highly
muscular pyloric sphincter, which controls movement of
food materials between the mid- and hindguts. Beyond
the sphincter, the tract expands into a distinctive cham-
ber, described as a fermentation chamber, which is lined
with cuticle and covered with distinctive lobe-like struc-
tures extending into the lumen from the cuticular surface.
The alimentary tract reverses direction before entering
the fermentation chamber, which consists of two large

C 2010 The Authors

sections lying on each side of the midgut. Interestingly,
the hindgut fermentation chamber (Fig. 1) is relatively
larger in the decaying wood and humus feeding larvae
of the scarabaeid subfamily Dynastinae than their root-
feeding counterparts in the subfamily Melolonthinae. On
leaving the fermentation tract the alimentary tract returns
to the anterior–posterior direction and extends into the
cuticle-lined rectum and finally the anus (Fig. 1).
Physicochemical properties of the scarab
larval gut
Gut pH
The alkaline midgut of scarabaeid larva differs from
most other coleopteran species. Biggs and McGregor
(1996) found the pH of the larval Costelytra zealandica
midgut and hindgut to be > 8, and that this varied depend-
ing on the location within these gut regions. In the pre-
caecal region the pH was 8.4 ± 0.69; it rose to 10.8 ± 1.28
in the anterior part of the remaining midgut, 10.9 ± 0.95 in
the middle of the midgut, and then dropped to 9.3 ± 0.68 at
the posterior end of the midgut. The pH of the hindgut was
8.2 ± 0.35 (Biggs & McGregor, 1996). Studies of Pachn-
oda ephippiata larvae also revealed a gut pH > 8. The pH
of the anterior midgut of P. ephippiata reached a maxi-
mum between 10.1 and 10.7, before declining to 8.4 ± 0.1
beyond the third row of caecae. The hindgut of first in-
star P. ephippiata larvae maintained a constant, slightly
alkaline pH value, although values could drop to neutral
toward the rectum in the second and third instars (Lemke
et al., 2003).The midgut of Pachnoda marginata similarly
is strongly alkaline with a pH of 9.5–11.7 (Cazemier et al.,
1997b). For Melolontha melolontha larvae the midgut pH
was fairly constant (pH 7.9–8.2), but increased to slightly
alkaline (pH 8.6) in the hindgut paunch before falling
again to neutral pH conditions in the colon and rectum
(Egert et al., 2005). Similar findings were recorded for
Sericesthis geminate larvae, where the midgut pH was 9.0
and hindgut pH was 7.5 (Soo Hoo & Dudzinski, 1967).
These observations show a consistent pattern among
the different scarab larvae with a high pH recorded in the
midgut, falling to a lower pH in the hindgut. This suggests
that the pH is maintained to assist the digestive process
through the scarab larval gut in a manner that facilitates
efficient absorption of nutrients (Terra & Ferreira, 1994;
Elpidina et al., 2001; Oppert et al., 2006). The alkaline
environment in the gut of scarab larvae has been proposed
as a way of helping to increase the solubility of organic
polymers in humus, assist with extraction of hemicellu-
loses from plant cell walls, and possibly aid the breaking

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The scarab gut – a bioreactor 177
up of stable organo-mineral complexes (Biggs & McGre-
gor, 1996; Kappler & Brune, 1999). Zhang and Brune
(2004) found that the proteolytic activities of the midgut
proteinases in P. ephippiata were higher at pH 12 than at
pH 7, and the midgut extract had higher activity in di-
gesting the proteinaceous component of the model humic
acid at pH 12 than at pH 7, indicating an adaptation of
proteinases to the physiological pH of the P. ephippiata
midgut. Thus, gut alkalinity may represent an important
nutritional adaptation for the uptake of essential or lim-
iting nutrients from plant roots and other organic matter
consumed by scarab larvae (Felton & Duffey, 1991).
Gut redox potential
Microbial activity partly determines physiological gut
conditions such as pH level, redox potential, and oxygen
concentration (Zimmer & Topp, 1998; Kappler & Brune,
2002). The oxygen status determines whether anaerobic
fermentation or aerobic oxidation of nutrients prevails
(Zimmer & Brune, 2005). For example, reducing con-
ditions are essential for the development of some strictly
anaerobic methanogenic bacteria found in the proctodeum
of Oryctes spp. (Bayon & Etiévant, 1980).
In Oryctes nasicornis larvae, the electric potential of the
midgut contents varied between +50 mV and −10 mV,
while the proctodeum (fermentation chamber) contents
ranged between −40 mV and −100 mV. Redox potential
values within these ranges are conducive to establishment
of a reducing environment within the intestine (Bayon
& Etiévant, 1980). In P. ephippiata, the midgut of first-
instar larvae favored oxidizing conditions; whereas the
redox potential shifted toward reducing conditions in lar-
vae of the second and third instars, demonstrating that
the redox potential can change considerably during larval
midgut development. However, reducing conditions pre-
vailed in the fermentation chamber across all larval instars
(∼ −100 ± 11 mV) (Lemke et al., 2003). Investigation of
the P. marginata larval digestive tract showed that redox
potentials in freshly prepared midguts and hindguts were
in the range of −100 to −200 mV, which is indicative of
an anaerobic environment (Cazemier et al., 2003). In con-
trast to P. marginata, the redox potential in the midgut of
M. melolontha larvae ranged from +220 to +340 mV, and
dropped sharply at the midgut–hindgut junction, to attain
a minimum (0 mV) in the anterior hindgut, and increased
again toward the rectum (Egert et al., 2005). Negative
redox potentials have also been detected in wood-feeding
cockroaches and termites, and in herbivorous lepidopteran
larvae (Veivers et al., 1982; Appel & Martin, 1990).
The chemical and enzymatic reactions associated with
digestion, absorption and detoxification of food in the
178 S. W. Huang et al.

insect gut are dynamic and sensitive to changes in pH,
redox potential and other physicochemical parameters
(Johnson & Felton, 1996a). Redox potential and pH af-
fected the oxidative state of tannins, phenolics and other
ingested compounds in the gut, as well as the activity of
digestive enzymes, by imposing physiological constraints
on the effectiveness of some classes of digestive enzymes
(Christeller et al., 1989; Johnson & Felton, 1996a, 1996b).
It is also found that redox conditions can alter the effects
of plant allelochemicals on insect herbivores (Appel &
Martin, 1990).
The significance of redox potential in the gut can
be illustrated through comparison with biodegradation
processes in the soil. Paar (1969) distinguished char-
acteristic stages of organic matter degradation in soil
that are associated with redox potential values. Val-
ues of less than +300 mV promoted the reducing en-
vironment required for degradation of organic matter
and accelerated the rate that soluble products could be
accumulated. As negative redox values were reached,
reducing conditions were strengthened and a strict anaer-
obic environment would prevail; at redox values be-
low −100 mV, carbohydrates were completely reduced
and methane could be generated. Thus, the redox val-
ues typically found in the hindgut of scarab larvae
(∼ −100 ± 11 mV) provide an environment that is com-
patible with the formation of methane. Similar physio-
chemical conditions exist in ruminants, except that the
redox potential in the stomach of ruminants reaches val-
ues in the order of −300 mV and the methane yield is
generally 20 times higher than for scarab insects (Bayon
& Etiévant, 1980; Lemke et al., 2003; Egert et al., 2005).
Gut microflora
Scarab larvae have a microbe-rich alimentary tract with
most organisms concentrated in the enlarged hindgut (fer-
mentation sac) (Fig. 2). The scarab hindgut has been con-
sidered analogous to the micro-organism-rich rumen of
higher mammals, which is the primary site of microbial
fermentation for digestion of plant organic material.
Previous studies of microbes in the hindgut of
C. zealandica revealed a dense population of flagellate
protozoa and bacteria, ranging from 2–5 × 1010 bacteria
per g wet weight of gut, with some of these microbes able
to digest pectin and xylan substrates (Bauchop & Clarke,
1975). Using plate counts of microbes and polymerase
chain reaction (PCR) – denaturing gradient gel elec-
trophoresis (DGGE) fingerprinting, Zhang and Jackson
(2008) further investigated the bacterial diversity within
the larval gut of C. zealandica. Results showed that num-
bers of cultured bacteria were significantly (P < 0.05)
higher in the hindgut than the midgut. The bacterial

Fig. 2 Features of the scarab hindgut and associated bacteria. (A) Thin section of a C. zealandica hindgut (also referred to as the
fermentation sac), showing the characteristic lobe-like structures. (B) Wet mount of a lobe from first instar C. zealandica larva
5 days post-eclosion observed under phase contrast microscopy. (C) Fluorescence image of the lobe from photo B after staining with
LIVE/DEAD Baclight (molecular probes); bacteria in the lobe appear green (SYTO9) and the hindgut cells appear red (propidium
iodide). (D) and (E) Scanning electron micrographs of the C. zealandica hindgut lobes displaying tree-like cuticular growths surrounded
by a cuticular meshwork. (F) Transmission electron micrograph of a cross section through the C. zealandica hindgut showing a portion
of the tree-like lobe structure (bottom left) and the diverse bacterial morphotypes. (G) Transmission electron micrograph showing
Clostridia-form bacteria from the hindgut lobes of C. zealandica.


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C Institute of Zoology, Chinese Academy of Sciences, Insect Science, 17, 175–183

community composition within the midgut was highly
variable and changed with site of collection or diet, while
the hindgut bacterial species community was less affected
by external factors and had more species diversity. The
dominant species identified by PCR-DGGE were affili-
ated with the Clostridiales with the predominant bacteria
affiliated to the genus Clostridium. The remaining bacte-
ria were aligned to the β-proteobacteria, δ-proteobacteria,
and Bacteroidetes. The research on bacterial community
profiles associated with the hindgut walls of individual
Dermolepida albohirtum third-instar larvae indicated that
a number of taxa were found consistently in all D. albo-
hirtum larvae. These taxa included representatives from
the “Endomicrobia”, Firmicutes, Proteobacteria and Acti-
nobacteria, and closely related hindgut bacteria found in
C. zealandica (Pittman et al., 2008).
By using terminal restriction fragment length polymor-
phism (T-RFLP) to analyze bacterial 16S rRNA genes
from gut samples, Egert et al. (2005) found that M.
melolontha midgut samples from individual larvae pos-
sessed a simple but variable and probably nonspecific
community structure. The hindgut samples were more di-
verse but highly similar, especially in the wall fraction,
which is suggestive of a gut-specific community involved
in digestion. Analysis of the microbiota from the hindgut
of M. melolontha showed it was dominated by clones re-
lated to the Clostridiales. Clones related to the Actinobac-
teria, Bacillales, Lactobacillales and γ -proteobacteria
were confined to the lumen, whereas clones related to the
β- and δ-proteobacteria were found only on the hindgut
wall, suggesting that specific bacterial species were re-
stricted to different compartments. Both the midgut and
hindgut had high acetate concentrations, indicating mi-
crobial fermentation was likely to occur in both compart-
ments.
Egert et al. (2003) studied the digestive tract of
P. ephippiata, and found these larvae also contain a com-
plex gut microbiota. The species composition differed
markedly between midgut and hindgut, and was clearly
distinct from the microbiota identified in the surround-
ing soil. Four fermentative metabolism bacteria groups
(Lactobacillales, Clostridiales, Bacillales and Cytophaga-
Flavobacterium-Bacteroides [CFB] phylum) were the
dominant phylogenetic groups identified from the gut
of P. ephippiata. 4 ,6-diamidino-2-phenylindole (DAPI)
counts showed cell densities of 8.9 ± 3.5 × 109 cells/g and
4.0 ± 1.4 × 1010 cells/g in the midgut and hindgut, respec-
tively. Based on 16S rRNA gene frequencies, Actinobac-
teria dominated the alkaline midgut, while the hindgut was
dominated by members of the CFB phylum. Investigation
of the related scarab, P. marginata, showed that the num-
bers of cultivable bacteria in the midgut and hindgut were

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C Institute of Zoology, Chinese Academy of Sciences, Insect Science, 17, 175–183
The scarab gut – a bioreactor 179
0.95 ± 0.3 × 109 /mL gut and 2.0 ± 0.47 × 1010 /mL gut
respectively, which was similar to P. ephippiata (Cazemier
et al., 1997a).
In vertebrates with rumen or hindgut fermentation that
relies almost completely on microbial activity for the di-
gestion of food, bacterial numbers in the intestinal tract
range up to 1011 /mL (Hungate, 1966). Comparable num-
bers of bacteria are also present in the hindgut of scarab
larvae (Cazemier et al., 1997a; Egert et al., 2003), sug-
gesting that these bacteria are important for food digestion
in scarabs (Cazemier et al., 1997a). In the gut of Pachn-
oda spp. larvae, the apparent dominance of fermenting
bacteria (i.e., Lactobacillales, Clostridiales, members of
the CFB phylum, and clones related to Turicibacter san-
guinis) correlates with the high lactate and acetate con-
centrations in hindgut (Lemke et al., 2003). Likewise,
the high acetate concentration in M. melolontha larval
hindguts indicates the presence of microbial fermentation
in this compartment, and methanogenesis was found to be
confined to the hindgut (Egert et al., 2005).
In C. zealandica, the most common bacterial species
were affiliated with the order Clostridiales (Zhang &
Jackson, 2008). Egert et al. (2005) found that the hindgut
of M. melolontha was also dominated by specific group-
ings of the Clostridiales. Even though the hindgut of
P. ephippiata was dominated by members of the CFB
phylum, a high proportion of Clostridiales was also iden-
tified in the hindgut by 16S rRNA gene sequence and
T-RFLP profile analysis (Egert et al., 2003). These obser-
vations seem to imply that members of the Clostridiales
have developed a symbiotic relationship with scarab lar-
vae (Zhang & Jackson, 2008).
(Hemi)cellulose digestion in the scarab gut
Lignocellulose is the predominant component of woody
plant material, as well as being the most abundant
form of biomass in terrestrial ecosystems. Fungi, bac-
teria and soil invertebrates are the major lignocellu-
lose decomposers (Ohkuma, 2003). Earlier studies have
demonstrated fibre digestion in some scarab beetle lar-
vae. Bayon and Mathelin (1980) injected 14 C-labelled
cellulose into isolated midgut and proctodeal segments
of O. nasicornis for in vitro incubation, and estimated
that 25%–39% of the ingested pure cellulose was de-
graded. By comparing the fibre content in the diet
and the feces, Cazemier et al. (1997b) demonstrated
that P. marginata digested 65% of neutral detergent fi-
bres present in their diets. Digestion of (hemi)cellulose
is thought to occur in the enlarged hindgut paunch
with the assistance of micro-organisms (Bayon, 1980;
180 S. W. Huang et al.
Martin, 1983; Cazemier et al., 2003; Zhang & Jackson,
2008; Douglas, 2009).
Because bacteria with (hemi)cellulolytic activity appear
to be absent in the midgut, it seems unlikely that microbial
degradation of cellulose and hemicellulose occurs in this
part of the intestinal tract (Cazemier et al., 2003). While
carboxymethyl cellulase (CMC-ase), β-glucosidase and
xylanase activities have been detected in the midgut of O.
nasicornis larvae and P. marginata, their activities were
low. In P. marginata, activity of CMC-ase in midgut ex-
tracts was 0.69 ± 0.23 U/mL gut, while β-glucosidase
activity was 0.28 ± 0.19 U/mL gut, and xylanase ac-
tivity was 0.18 ± 0.11 U/mL gut. All three activities
were one to two orders of magnitude lower than the
other wood-feeding arthropods examined (Pycnoscelus
surinamensis, Mastotermes darwiniensis, Hylotrupes ba-
jules, etc.) (Cazemier et al., 1997b). Furthermore, it is
not clear whether these enzymes are functional under the
alkaline conditions of the midgut (Bayon & Mathelin,
1980; Cazemier et al., 1997b, 2003). Therefore, it has
been proposed that the midgut likely serves a predigestive
function for lignocellulose digestion, with the anaerobic
conditions of the hindgut facilitating processes required
for further biodegradation of the lignocellulosic material
(Cazemier et al., 2003; Zverlov et al., 2003).
Based on the high number of fermentative bacte-
ria present in the hindgut of scarab larvae, the CMC-
ase activity 0.29 ± 0.35 U/mL gut, β-glucosidase ac-
tivity 0.13 ± 0.09 U/mL gut and xylanase activity
0.6 ± 0.4 U/mL gut in hindgut extracts of P. marginata
(Cazemier et al., 1997b), and the existence of cellulolysis
in intestinal contents of proctodeal segments of O. nasi-
cornis larvae (demonstrated by injection of 14 C-cellulose
into the gut; Bayon & Mathelin, 1980), it has been sug-
gested that these intestinal bacteria were responsible for
cellulose degradation (Bayon & Mathelin, 1980; Martin
et al., 1991; Cazemier et al., 1997b; Zhang & Jackson,
2008). As noted above, Clostridiales is the dominant bac-
teria within the hindgut of C. zealandica and M. melolon-
tha larvae (Egert et al., 2003, 2005; Zhang & Jackson,
2008). Since the genus Clostridium contains a wide range
of species able to ferment sugars and more complex
molecules including cellulose, hemicellulose, pectin and
polysaccharides (Hippe et al., 1992; Varel et al., 1995;
Weber et al., 2001; Zhang & Jackson, 2008), it seems
likely that the Clostridium spp. identified from scarab
hindguts have an important role in the degradation of the
roots and organic matter consumed by scarab larvae (Egert
et al., 2005; Zhang & Jackson, 2008).
The anaerobic conditions and production of methane
in the fermentation chamber of scarab larvae parallels
digestion in ruminants. The breakdown of cellulose by
Journal compilation 
C Institute of Zoology, Chinese Academy of Sciences, Insect Science, 17, 175–183
extracellular symbiotic organisms leads to the forma-
tion of volatile fatty acids (VFA), such as acetic acid
(Bayon & Mathelin, 1980) and by-products that can act as
substrates for methanogenic bacteria (Bayon & Etiévant,
1980). The diffusion of small molecules, such as acetic
acid, is uninhibited in structures involved in absorption
and the cuticlar membrane. The production of methane
took place exclusively in the hindgut of O. nasicornis
larvae (Bayon, 1980). Methanogens were closely associ-
ated with the chitinous lobe-like structures formed within
the host’s fermentation sac in the hindgut of P. ephippiata
(Egert et al., 2003). In addition, accumulation of microbial
fermentation products had been detected in the hindgut of
P. ephippiata larvae (Lemke et al., 2003). These exper-
iments demonstrated that the high level of cellulolytic
activity was associated with bacteria in the hindgut of
scarab larvae.
In fact, as early as 1926, a cellulolytic bacterial species,
Bacillus cellulosam fermentans, was isolated from en-
richment cultures inoculated with hindgut contents of a
rose chafer, Potosia cuprea; unfortunately specific exper-
iments demonstrating in vivo cellulolytic activity were
not conducted for this bacterium (Werner, 1926). How-
ever, a number of facultative anaerobic and strictly anaer-
obic bacteria with (hemi)cellulolytic activity have been
isolated from the hindgut of P. marginata (Cazemier
et al., 2003). The dominant (hemi)cellulolytic species
was Promicromonospora pachnodae, which can produce
xylanase and endoglucanase activities against several
plant-derived polymers under both aerobic and anaerobic
conditions. Moreover, the (hemi)cellulolytic bacterium,
Cellulomonas pachnodae, was isolated from the hindgut
of P. marginata larvae, and two new xylanase-encoding
genes, named xyn11A and xyn10B, were isolated from
a genomic library and expressed in Escherichia coli
(Cazemier et al., 1997b, 1999, 2003). Geissinger et al.
(2009) isolated Elusimicrobium minutum, the first culti-
vated representative of the termite group 1 phylum from a
humivorous scarab beetle larva. It can grow heterotroph-
ically on sugars, and is able to ferment D-galactose,
D-glucose, D-fructose, D-glucosamine, and N-acetyl-D-
glucosamine to acetate, ethanol, hydrogen, and alanine as
major products (Geissinger et al., 2009).
The successful isolation of bacteria with
(hemi)cellulolytic activity from scarab hindguts
shows that micro-organisms participate in the digestion
of cellulose. The absence of cellulases from hindgut fluid
suggests that cellulose digestion is due to the activity
of cell-bound enzymes produced by the bacteria, rather
than from secreted extracellular enzymes (Martin, 1983).
Indeed, bacteria possessing (hemi)cellulolytic activity
have been found attached to the fragments of plant tissue

C 2010 The Authors

present within scarab hindguts (Martin, 1983). The
final product of cellulose degradation is mainly acetic
acid, which appears to pass through the hindgut wall for
subsequent utilization by the insect (Bayon & Mathelin,
1980).
With the identification and isolation of bacteria pos-
sessing cellulolytic and hemicellulolytic activities from
scarab digestive tracts, a new source of (hemi)cellulolytic
enzymes has been established. Future work will no doubt
characterize specific cellulase and hemicellulase genes
and enzymes, such as xylanases and endoglucanases, from
bacteria identified from scarab hindguts (Cazemier et al.,
1999), and this research will help develop the potential
of scarab-associated (hemi)cellulolytic activities for use
within the bio-fuel industry.
Conclusion
Larvae from the Scarabaeidae family consume soil, wood
and organic matter, the nutrients of which are extracted
with the assistance of digestive enzymes present within
the mid- and hindgut. The hindgut is the main region
for digestion of (hemi)celluloses in scarab larvae as evi-
denced by high concentrations of volatile fatty acids, the
presence of fermenting bacteria and the occurrence of typ-
ical anaerobic activity, such as methanogenesis (Bayon,
1980; Brune, 1998; Egert et al., 2003). (Hemi)cellulose
digestion is probably mediated by hindgut bacteria
(Werner, 1926; Cazemier et al., 1997b), and the ex-
panded hindgut structure with its modified lobes appears
to have evolved to function as a refuge for these sym-
biotic microbes. Novel (hemi)cellulolytic bacteria, such
as P. pachnodae and E. minutum have now been isolated
from the larvae hindgut of scarab larvae (Cazemier et al.,
2003; Geissinger et al., 2009). These findings indicate
that the larval scarab gut is not only a fruitful source of
microbial biodiversity and functional novelty, but also a
new source of micro-organisms and enzyme activities that
will aid the function and design of future bioreactors for
the bio-fuel industry.
Acknowledgments
This research was supported by National Natural Science
Foundation of China (30671404), the earmarked fund for
Modern Agro-industry Technology Research System of
China, the Research Fund for the Doctoral Program of
Higher Education of China (200805040010) and the New
Zealand Foundation for Research Science and Technol-
ogy. The authors would also like to thank Sue Zydenbos
for editorial assistance.

C 2010 The Authors
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The scarab gut – a bioreactor 181
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Accepted December 11, 2009
 Insect Science (2010) 17, 184–198, DOI 10.1111/j.1744-7917.2010.01322.x

REVIEW

Methods for discovery and characterization of cellulolytic

enzymes from insects

Jonathan D. Willis, Cris Oppert and Juan L. Jurat-Fuentes

Department of Entomology and Plant Pathology, University of Tennessee, Knoxville, Tennessee, USA

Abstract Cellulosic ethanol has been identified as a crucial biofuel resource due to its
sustainability and abundance of cellulose feedstocks. However, current methods to obtain
glucose from lignocellulosic biomass are ineffective due to recalcitrance of plant biomass.
Insects have evolved endogenous and symbiotic enzymes to efficiently use lignocellulosic
material as a source of metabolic glucose. Even though traditional biochemical methods
have been used to identify and characterize these enzymes, the advancement of genomic
and proteomic research tools are expected to allow new insights into insect digestion of
cellulose. This information is highly relevant to the design of improved industrial processes
of biofuel production and to identify potential new targets for development of insecticides.
This review describes the diverse methodologies used to detect, quantify, purify, clone and
express cellulolytic enzymes from insects, as well as their advantages and limitations.
Key words biofuels, cellulases, cellulase discovery methods, cellulase substrate, insect
digestive fluids, lignocellulosic biomass

Insect relevance to lignocellulosic biofuels
Current world energy needs demand the development of
industrial-scale processes for the sustainable production
of fuel from renewable biological resources as economic
and environmentally sound alternatives to finite fossil
fuels. In the US, lignocellulosic ethanol has been sug-
gested as a desirable biofuel, mostly due to its sustain-
ability, reduced competition as a food resource, net en-
ergy production, and reduced input costs related to pro-
duction of ethanol from corn-derived starch (Lynd et al.,
1991; McLaughlin et al., 2002; Schmer et al., 2008).
Cost-efficient production of ethanol from lignocellulosic
biomass is mostly dependent on development of efficient
hydrolysis technologies (Sun & Cheng, 2002; Wyman,
2007). Enzymatic degradation of cellulose is considered
the hydrolysis method with the greatest potential for im-
provement and cost reduction (Wyman, 1999, 2007). Cur-
Correspondence: Juan Luis Jurat-Fuentes, Department of
Entomology and Plant Pathology, University of Tennessee,
Knoxville, TN 37996-4560, USA. Tel: (865) 974 5931; email:
jurat@utk.edu
rent estimates suggest that reducing the cellulase enzyme
amounts by half through biotechnology could decrease
processing costs by up to 13% (Lynd et al., 2008).
Lignocellulosic recalcitrance, which prevents enzy-
matic access to fermentable sugars, is derived from the
tight association of cellulose with hemicellulose and
lignin to form the plant cell wall (Delmer & Amor, 1995).
Pre-treatment steps are currently necessary to achieve
efficient glucose yields from lignocellulosic feedstocks
(Chandra et al., 2007). Even though novel pretreatment
technologies based on ionic liquids (Zhao et al., 2009)
or expression of hydrolases in plants (Taylor et al., 2008)
are being developed, there is still a need to also iden-
tify and develop more efficient cellulolytic enzymes that
can be applied into both pretreatment and/or cellulolytic
technologies (Mosier et al., 2005).
Cellulose degradation requires the synergistic ac-
tion of three types of glycoside hydrolases (GH):
endo-β-1,4-glucanases (EG; EC. 3.2.1.4), exo-β-1,
4-cellobiohydrolases (CBH; EC. 3.2.1.91), and β-
glucosidases (EC. 3.2.1.21) (Clarke, 1997). EG enzymes
work by random cleavage of β-1,4 glycosidic bonds in the
internal portions of cellulose strands to reduce the degree


C 2010 The Authors 184
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C Institute of Zoology, Chinese Academy of Sciences

of polymerization of the cellulose chain into smaller sub-
units. CBH enzymes remove subunits at both reducing
and non-reducing ends of the cellulose chain, releasing
either cellobiose or glucose. Due to the inhibition of EG
enzymes by accumulation of cellobiose, the presence of
β-glucosidases to hydrolyze cellobiose to glucose is im-
portant for complete degradation of cellulose (Holtzapple
et al., 1990; Gruno et al., 2004).
Enzymes currently used for production of cellu-
losic ethanol were identified in fungal and bacterial
systems (Lynd, 1996). Current limitations of enzy-
matic degradation of lignocellulosic biomass are mostly
related to enzymatic stability and susceptibility to
inhibitory agents or byproducts (Mousdale, 2008;
Kristensen et al., 2009). Continuous prospecting and bio-
engineering efforts should provide novel enzymes with
higher specific activity and with lower susceptibility to
inhibitors (Lynd et al., 2008).
Some insects have evolved very effective strategies
to use lignocellulosic substrates as sources of energy
(Martin, 1983), which makes them an optimal resource
to prospect for novel cellulolytic enzymes. Evidence for
the prospecting potential in insects is most notably found
in species of termites, which are known to live almost ex-
clusively on substrates with high lignocellulosic content.
In lower termites cellulolytic activity is mostly dependent
on enzymes produced by symbiotic protozoa (Ohkuma,
2008), while higher termites combine cellulases secreted
by the gut cells with bacterial enzymes (Tokuda et al.,
1997; Warnecke et al., 2007). The importance of insect-
produced cellulases for survival has also been suggested
as a potential target for development of termite control
technologies (Zhu et al., 2005; Zhou et al., 2008). In-
creased thermostability and specific activity of termite
cellulases achieved through random mutagenesis of non-
conserved residues highlights the potential development
of technologies for biofuel production from these insect
enzymes (Ni et al., 2007). Recent evidence also suggests
that lignin degradation, which is one of the main issues of
plant biomass use for biofuels, may be common in insects
feeding on lignocellulosic substrates (Geib et al., 2008).
In comparison to extensive studies in termite cellulolytic
systems, detailed information on alternative insect cel-
lulolytic systems is limited. This review focuses on the
methods reported to detect and characterize cellulolytic
systems in insects, with emphasis on the insect-produced
enzymes.
Discovery of insect cellulases
Based on data from termites and wood-feeding roaches,
cellulolytic activity in insects has been attributed to sym-

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C Institute of Zoology, Chinese Academy of Sciences, Insect Science, 17, 184–198
Methods for insect cellulases 185
biotic gut flora (Cleveland, 1924, 1934). The role of sym-
biotic microbes in production of cellulolytic enzymes has
been widely recognized for insects feeding on lignocel-
lulosic biomass (Martin, 1983; Morrison et al., 2009).
However, examples of insect-produced cellulolytic activ-
ity have been described for species belonging to five tax-
onomic orders: Isoptera (Martin & Martin, 1978; Slaytor,
1992; Bignell et al., 1994), Thysanura (Treves &
Martin, 1994), Coleoptera (Genta et al., 2006), Blattodea
(Scrivener et al., 1989; Genta et al., 2003) and Hemiptera
(Adams & Drew 1965). Low numbers of bacteria in gut re-
gions that display cellulolytic activity has been suggested
as evidence for endogenous cellulase degradation in some
species of Orthoptera and Phasmatodea (Cazemier et al.,
1997).
The first insect cellulase gene, encoding an endo-β-
1,4-glucanase, was cloned from Reticulitermes speratus
(Watanabe et al., 1998). While no endogenous CBHs have
been reported in insects, EG genes have been identified
in other isopteran species (Tokuda et al., 1999; Scharf
et al., 2005; Zhang et al., 2009a), as well as species in
the orders Coleoptera (Ferreira et al., 2001; Sugimura
et al., 2003; Lee et al., 2004, 2005; Wei et al., 2006b) and
Orthoptera (Kim et al., 2008). Availability of sequenced
genomes is allowing a more complete identification and
characterization of insect cellulolytic systems (Kunieda
et al., 2006). Sequenced insect EGs have been included in
diverse GH families based on sequence homology, includ-
ing GHF1, GHF5, GHF7, GHF9, GHF10 and GHF45. The
only three-dimensional structure for an insect cellulase,
the NtEgl endo-glucanase from Nasuritermes takasagoen-
sis, revealed the common alpha helical barrel folding and
catalytic domain observed for GHF9 members (Khademi
et al., 2002).
Quantitative and qualitative detection
of cellulolytic activity in insects
Detection of enzymatic activity is directly dependent on
the sample preparation as well as the specific substrate
used. Due to their involvement in digestion, salivary
glands, gut tissues and gut digestive fluids are usually used
for the detection of cellulolytic activity in insects (Martin,
1983; Cazemier et al., 1997), and due to localized expres-
sion, levels of activity or cellulases detected are greatly
dependent on the tissue of origin or sample preparation
method (Martin, 1983; Ferreira et al., 2002). Another im-
portant consideration for the quantification of cellulolytic
activity is the product inhibition reported for both EG and
CBH activities (Zhang et al., 2009b). This limitation is
generally overcome by addition of β-glucosidases to the
reaction to degrade inhibiting cellobiose to glucose.
186 J. D. Willis et al.

In most cases, degradation of cellulase substrates is
measured using modifications of the 3,5-dinitrosalicylic
acid (DNSA) (Miller, 1959) or tetrazolium blue (Jue
& Lipke, 1985) assays, which detect reducing sug-
ars generated during cellulose degradation. Initially, the
DNSA assay was found susceptible to interferences when
using native or pretreated lignocellulosic materials, which
limited its use depending on the substrates (Rivers et al.,
1984). More recently, optimized microplate assays based
on the DNSA method to quantify degradation of mod-
ified cellulose (Xiao et al., 2005), filter paper (FP)
(Xiao et al., 2004), or pretreated lignocellulosic mate-
rial (King et al., 2009), have been developed to overcome
these limitations. Other techniques to quantify produc-
tion of glucose in solution include the alkaline copper
(Somogyi, 1952) and the glucose oxidase/peroxidase
(Dahlqvist, 1968) methods. However, the oxidase method
has been shown to be affected by the presence of
lignin (Breuil & Saddler, 1985), and although it is
rarely used for lignocellulosic substrates, it has been
used with more processed substrates to determine β-
glucosidase activity in insect samples (Ferreira & Terra,
1983; Chipoulet & Chararas, 1985a; Zinkler & Gotze,
1987; Marana et al., 1995, 2000; Yapi et al., 2009).
Methods based on HPLC or thin layer chromatogra-
phy separations to estimate produced sugars have also
been described (Adams & Drew, 1965; Berlin et al.,
2006; Chundawat et al., 2008). To further character-
ize the activity of specific cellulolytic enzymes, activ-
ity is measured by spectrophotometry using substrates
containing p-nitrophenol (Terra et al., 1979; Chipoulet
& Chararas, 1985a; Cazemier et al., 1997; Marana
et al., 2000; Ferreira et al., 2001; Marana et al., 2004;
Yapi et al., 2009) or methyl-umbelliferyl (MU) groups
(Jacobson & Schlein, 1997; Marana et al., 2001).
Use of diverse cellulase substrates can help differenti-
ate the specific contribution of specific insect gut areas
to the digestion of plant material (Tokuda et al., 2005),
which can contribute to a more complete characteriza-
tion of the cellulolytic process. Due to their low levels
of chemical modification, FP, microcrystalline cellulose
(MCC) and cotton, have been used as preferred cellulase
substrates to determine the existence of complete cel-
lulolytic systems (EG, CBH and β-glucosidases). How-
ever, there is evidence for the degradation of MCC in
insects lacking CBH activity (Scrivener & Slaytor, 1994).
As shown in Table 1, reports of CBH activity in in-
sects are uncommon (Cazemier et al., 1997), and this
activity is related to enzymes produced by symbionts or
parasites (Martin & Martin, 1978; Martin, 1983). The
main limitation in the use of these CBH substrates is
their insolubility, which limits their use to in-solution as-
says with continuous mixing and complicates the removal
of particulates. A high throughput microplate assay for

Table 1 Insects with reported cellobiohydrolases and the methods used for the quantitative detection of this activity.

Order (family) Species Substrate Detection method Reference

Coleoptera Anoplophora glabripennis MCC DNSA Li et al., 2008

(Cerambycidae)
Rhagium inquisitor MCC AC Chipoulet & Chararas, 1985b
Diptera (Psychodidae) Phlebotomus papatasi MeUMB Flu Jacobson & Schlein, 1997
Isoptera Coptotermes lacteus MCC TB Hogan et al., 1988
(Heterotermitidae)
(Kalotermitidae) Neotermes koshunensis MCC TB Tokuda et al., 2005
(Rhinotermitidae) Reticulitermes flavipes pNPC Abs. Zhou et al., 2007
R. speratus, Coptotermes MCC TB Tokuda et al., 2005
formosanus
(Termitidae) Odontotermes formosanus, MCC TB Tokuda et al., 2005; Tokuda
Nasutitermes & Watanabe, 2007
takasagoensis
(Termopsidae) Hodotermopsis sjoestedti MCC TB Tokuda et al., 2005
Thysanura Thermobia domestica MCC GOP Zinkler & Gotze, 1987
(Lepismatidae)

AC, alkaline copper; Abs, absorbance; DNSA, dinitrosalicylic acid; Flu, fluorescence; GOP, glucose oxidase-peroxidase; MCC, micro-
crystalline cellulose; MeUMB, 4-methylumbelliferyl-β-cellobiopyranoside; pNPC, p-nitrophenyl-β-D-cellobioside, TB, tetrazolium
blue.


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C Institute of Zoology, Chinese Academy of Sciences, Insect Science, 17, 184–198

cellulolytic activity was developed using MCC as sub-
strate (Chundawat et al., 2008), although this approach
has not been used with insect samples. An alternative
approach is the detection by absorbance or fluorescence
of p-nitrophenol or methyl-umbelliferyl (MU) cleavage
after hydrolysis of glycosides such as p-nitrophenyl-
β-D-cellobioside (pNPC) (Zhou et al., 2007), or
4-methylumbelliferyl-β-cellobiopyranoside (MeUMB)
(Jacobson & Schlein, 1997). In termites it has been sug-
gested that localization of activity against MCC correlates
with the presence of hindgut symbionts (Tokuda et al.,
2005). The same cellulase substrate was also used to qual-
itatively determine the existence of cellulolytic activity
in gut symbionts isolated from three coleopteran species
(Delalibera et al., 2005) or to establish the role of
fungal cells for cellulolytic activity in fungus-growing
termites (Abo-Khatwa, 1978). A similar direct cor-
relation between activity against FP and numbers of
hindgut symbionts was reported in Periplaneta americana
(Gijzen et al., 1994). Survival and assimilation of glucose
from FP digestion by Panesthia cribrata in the presence
of antibiotics was used as evidence for the presence of
endogenous complete cellulolytic systems in this insect
(Scrivener et al., 1989).
An alternative to insoluble cellulase substrates are mod-
ified celluloses, such as carboxymethylcellulose (CMC),
which are derived to improve water solubility. In CMC the
hydroxyl groups are methylated, resulting in high water
solubility compared to crystalline or amorphous cellulose.
Due to its ease of use and easy degradation by EG activity,
CMC is the most documented cellulase substrate used for
solution assays or incorporation into agar or acrylamide
gel matrices (Table 2). As indicated in Table 2, degradation
of CMC quantified by the DNSA assay is the most com-
mon technique used to demonstrate cellulolytic activity in
insect samples. CMC has been used as substrate in agarose
plates for qualitative determinations of EG activity lo-
calization in gut regions of Rhagium inquisitor (Zverlov
et al., 2003), as early screening for cellulolytic symbionts
(Delalibera et al., 2005), and to screen modified (Ni et al.,
2007; Zhang et al., 2009a) or heterologously produced in-
sect cellulases (Lee et al., 2004; Ni et al., 2005; Wei et al.,
2005, 2006b). In this method, plates are incubated with
digestive fluids and activity revealed as clear zones when
staining undigested CMC with Congo red dye. The di-
ameter of this activity area has been utilized as a relative
measurement to compare activity of heterologously ex-
pressed enzymes from Coptotermes formosanus (Zhang
et al., 2009a). In a similar strategy, cellulolytic zymograms
with CMC as substrate can be used to detect proteins
with cellulolytic activity after electrophoretic separation

C 2010 The Authors
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C Institute of Zoology, Chinese Academy of Sciences, Insect Science, 17, 184–198
Methods for insect cellulases 187
of proteins (Schwarz et al., 1987). The advantage of this
method over activity assays in agar plates is that specific
protein bands with cellulolytic activity can be visualized
and their molecular weight estimated. To prevent CMC
degradation during electrophoresis, proteins are only par-
tially heat denatured and gels run at low temperature so
that discrete activity bands, rather than smears, can be de-
tected. Using this technique, specific cellulases have been
detected from digestive fluids of diverse insects, includ-
ing R. inquisitor (Zverlov et al., 2003), T. molitor (Genta
et al., 2006), Psacothea hilaris (Sugimura et al., 2003),
Anoplophora glabripennis (Li et al., 2008), and Phaedon
cochleariae (Girard & Jouanin, 1999). These zymograms
have also been used to characterize cellulolytic activity of
insect cellulases overproduced in heterologous systems
(Ni et al., 2005; Zhang et al., 2009a).
Due to the diverse range of specificities of
β-glucosidases, a variety of substrates are used for de-
tecting and quantifying this enzymatic activity. Cleav-
age of β-D-glucopyranosides (such as salicin, octyl β-
glucoside or helicin), or disaccharides (such as cel-
lobiose or amygdalin) by β-glucosidases is generally
quantified by detection of the generated glucose us-
ing the glucose oxidase/peroxidase method (Ferreira
& Terra, 1983; Chipoulet & Chararas, 1985a; Zinkler
& Gotze, 1987; Marana et al., 1995; Marana et al.,
2000; Yapi et al., 2009). Alternatively, β-glucosidase
activity is also quantified by measuring the hydrol-
ysis of p-nitrophenol from glycoside derivatives (NP
β-glycosides) such as glucoside (NP βGlu) (Terra et al.,
1979; Chipoulet & Chararas, 1985a; Cazemier et al.,
1997; Marana et al., 2000; Ferreira et al., 2001; Marana
et al., 2004; Yapi et al., 2009), or methyl-umbelliferyl
(MU) fluorescence after hydrolysis of MU glycosides
such as 4-methyl-umbelliferyl β-D-glucoside (MUG)
(Marana et al., 2001). Activity properties and speci-
ficity of purified β-glucosidases from digestive systems
of lepidopteran (Marana et al., 2000, 2001), coleopteran
(Chipoulet & Chararas, 1985a, 1985b; Ferreira & Terra,
1989; Genta et al., 2006), orthopteran (Marana et al.,
1995), and dipteran (Terra et al., 1979; Ferreira & Terra,
1983) insects have been reported using these meth-
ods. Table 3 provides a comprehensive list of insects
prospected for β-glucosidases and the specific substrate
and methodology used. Combined use of diverse sub-
strates allowed determination of enzymatic specificities
in insect midgut β-glucosidases from diverse taxonomic
groups (Ferreira et al., 1998). Protein bands display-
ing β-glucosidase activity after electrophoretic separation
can be detected using MUG as substrate (Genta et al.,
2006).
188 J. D. Willis et al.

Table 2 Insects with documented β-1,4-endoglucanase activity and the methods used for the quantitative detection and characterization
of this activity. When available, information on the enzyme purification and molecular size are also presented. NP = not provided by
the authors.

Detection Size
Order (family) Species Substrate Purification Reference
method (kDa)

Blattodea Panesthia cribrata CMC DNSA SEC, AEC, 53.6, Scrivener & Slaytor,
(Blaberidae) HIC 48.8 1994
Pycnoscelus surinamensi CMC DNSA NP NP Cazemier et al., 1997
(Blattidae) Blaberus fuscus, CMC DNSA NP NP Cazemier et al., 1997;
Periplaneta americana, Gijzen et al., 1994
P. australasia
Coleoptera Agrilus planipennis CMC CMC-AP NP NP Vasanthakumar et al.,
(Buprestidae) 2008
(Cerambycidae) Anoplophora glabripennis CMC DNSA, NP NP Li et al., 2008
Zymogram
Apriona germari CMC DNSA, SEC, AEC 29, Lee et al., 2004; Lee
CMC-AP 36, et al., 2005; Wei
47 et al., 2006b
Hylotrypus bajules CMC DNSA NP NP Cazemier et al., 1997
Psacothera hilaries CMC TB Native 47 Sugimura et al., 2003
PAGE
Rhagium inquisitor CMC AC NP NP Chipoulet & Chararas,
1985b
Saperda vestita CMC CMC-AP NP NP Delalibera et al., 2005
(Chrysomelidae) Aulacophora foveicollis CMC Zymogram, Native NR Sami & Shakoori, 2008
DNSA PAGE
(Curculionidae) Dendroctonus frontalis CMC CMC-AP NP NP Delalibera et al., 2005
Ips pini CMC CMC-AP NP NP (Delalibera et al.,
2005)
(Scarabeidae) Pachnoda marginata CMC DNSA NP NP (Cazemier et al., 1997)
Diptera Phlebotomus papatasi OBRH, CA Abs. NP NP (Jacobson & Schlein
(Psychodidae) 1997)
(Sciaridae) Rhynchosciara americana CMC Colorimetry NP NP (Terra et al., 1979)
(Tipulidae) Tipula abdominalis CMC DNSA NP NP (Walters & Smock
1991)
Isoptera Coptotermes lacteus CMC TB NP NP (Hogan et al., 1988)
(Heterotermitidae)
(Kalotermitidae) Neotermes koshunensis CMC TB NP NP (Tokuda et al., 2005)
Cryptotermes CMC AC NP NP (Mo et al., 2004)
pingyangensis
(Mastotermitidae) Mastotermes darwiniensi CMC Zymogram, AEC 36 (Cazemier et al., 1997;
DNSA Li et al., 2003)
(Rhinotermitidae) Reticulitermes flaviceps CMC DNSA, AC NP NP (Mo et al., 2004; Zhou
et al., 2007)
R. speratus CMC TB NP NP (Watanabe et al., 1998)
Coptotermes formosanus, CMC AC NP NP (Mo et al., 2004)
R. leptomandibularis

Continued


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 Methods for insect cellulases 189

Table 2 Continued

Detection Size
Order (family) Species Substrate Purification Reference
method (kDa)

(Termitidae) Nasutitermes takasagoensis CMC TB, SEC 47 Tokuda & Watanabe,

Zymogram 2007; Tokuda et al.,
1997
Odontotermes formosanus CMC AC AEC 80 Mo et al., 2004;
Yang et al., 2004
Lepidoptera Parnassius apollo ssp. CMC DNSA NP NP Nakonieczny et al.,
(Papilionidae) frankenbergeri 2006
(Saturniidae) Philosamia ricini CMC AC NP NP Pant & Ramana, 1989
Orthoptera Schistocerca gregaria CMC DNSA NP NP Cazemier et al., 1997
(Acrididae)
(Gryllidae) Acheata domesticus CMC DNSA NP NP Cazemier et al., 1997
Teleogryllus emma CMC DNSA TAC 47 Kim et al., 2008
Plecoptera Peltoperla arcuata CMC AC NP NP Walters & Smock,
(Peltoperlidae) 1991
(Pternoarcidae) Allonarcys proteus CMC AC NP NP Walters & Smock,
1991
Phasmatodea Eurycantha calcarata CMC DNSA NP NP Cazemier et al., 1997
(Phasmatidae)
Trichoptera Pycnopsyche spp. CMC AC NP NP Walters & Smock,
(Limnephilidae) 1991
Thysanura Thermobia domestica CMC GOP NP NP Zinkler & Gotze, 1987
(Lepismatidae)
Ctenolepisma lineata RC AC NP NP Lasker & Giese, 1956

Abs, Absorbance; AEC, anion exchange chromatography; AC, alkaline copper; AP, agarose plates; CA, cellulose azure; CMC, car-
boxymethylcellulose; DNSA, dinitrosalicylic acid; HIC, hydrophobic interaction chromatography; NP, not provided; OBRH, ostazin
brilliant red hydroxyethyl-cellulose; RC, regenerated cellulose; SEC, size exclusion chromatography; TAC, tag affinity chromatography;
TB, Tetrazolium blue.

Identification, cloning and expression of insect
cellulases
In order to characterize specificity, substrate affinity and
activity, detected insect cellulases need to be either pu-
rified or cloned and expressed heterologously. As in the
case of fungal and bacterial cellulases, insect cellulases
are usually not expressed at high levels, hindering their
characterization through purification efforts. Probably re-
flecting their abundance over other glycosidases, most
purified insect cellulases are β-glucosidases. Purification
procedures usually consist of multiple steps, including
size exclusion, anion exchange and/or hydrophobic inter-
action chromatography (Marana et al., 1995, 2000; Fer-
reira et al., 2001; Yapi et al., 2009). Even though proteins
displaying CBH activity have not been purified from in-
sect systems, EG enzymes have been purified and char-
acterized from cockroach species (Scrivener & Slaytor,
1994; Genta et al., 2003) or termite symbiotic flagel-
lates (Li et al., 2003) using also liquid chromatographic
procedures. Alternative reported purification methods
for glycosidases include isoelectric focusing (Ferreira &
Terra, 1983) and preparative electrophoresis (Chipoulet &
Chararas, 1985a; Sugimura et al., 2003; Sami & Shakoori,
2008).
Once glycosidases are purified, protein sequencing may
facilitate primer design for polymerase chain reaction
(PCR) cloning (Marana et al., 2001; Sugimura et al.,
2003). A disadvantage to this cloning method is primer de-
generacy, which may result in lack of specific amplicons
from PCR reactions. Additionally, selection of template
material may be difficult if no information on the origin
(insect vs. symbiont) of the enzyme is available. Probably
due to these limitations of PCR cloning, the most reported
method to clone and sequence insect cellulases is the gen-
eration and screening of cDNA libraries. In the case of


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190 J. D. Willis et al.

Table 3 Insects with documented β-D-glucosidase and the methods used for the quantitative detection and characterization of this
activity. When available, information on the enzyme purification and molecular size are also presented. NP = not provided by the
authors.

Detection Purifi- Size

Order (family) Species Substrate Reference
method cation (kDa)

Blattodea Panesthia cribrata pNPG Abs. NP NP Scrivener &

(Blaberidae) Slaytor, 1994
Gromphadorrhinna pNPG Abs. NP NP Cazemier et al.,
portentosa 1997
(Blattidae) Blaberus fuscus pNPG Abs. NP NP Cazemier et al.,
Periplaneta 1997
americana P.
australasia
(Blattodea) Pycnoscelus pNPG Abs. NP NP Cazemier et al.,
surinamensi 1997
Coleoptera Pheropsophus CB, pNPG, GOP DC 27 Ferreira & Terra,
(Carabidae) aequinoctialis amygdalin, 1989; Ferreira
salicin et al., 1998
(Cerambycidae) Anoplophora Salicin DNSA NP NP Li et al., 2008
glabripennis
Hylotrypus bajules pNPG Abs. NP NP Cazemier et al.,
1997
Rhagium inquisitor CB, lactose, GOP, Abs. DC, PE NR Chipoulet &
gentiobiose, Chararas,
maltose, 1985a, 1985b
pNPG, salicin
(Curcolinidae) Rhynchophorus pNPG Abs. SEC, 58 Yapi et al., 2009
palmarum AEC,
HIC
(Elateridae) Pyrearinus pNPG, CB, Abs., GOP NP NP Ferreira et al.,
termitilluminans salicin, 1998
amygdalin
(Scarabeidae) Pachnoda pNPG Abs. NP NP Cazemier et al.,
marginata 1997
(Tenebrionidae) Tenebrio molitor pNPG, MUG, Abs., PE 59 Ferreira et al.,
CMC, CB, zymo- 1998, 2001
amygdalin, grams,
salicin GOP
Diptera Ptychoptera MUG Flu NP NP Wolf et al., 1997
(Nematocera) paludosa
(Sciaridae) Rhynchosciara pNPG, CB, Flu, GOP DGC 106 Ferreira & Terra,
americana maltose, salicin and 1983; Ferreira
65 et al., 1998;
Terra et al.,
1979
Isoptera Coptotermes lacteus CB TB NP NP Hogan et al.,
(Heterotermitidae) 1988

Continued


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 Methods for insect cellulases 191

Table 3 Continued

Detection Purifi- Size

Order (family) Species Substrate Reference
method cation (kDa)

(Kalotermitidae) Cryptotermes Salicin AC NP NP Mo et al., 2004

pingyangensis
Neotermes CB GOM Recom- 60 Ni et al., 2007
koshunensis binant
(Macrotermitdidae) Odontotermes Salicin AC NP NP Mo et al., 2004
formosanus
(Mastotermitidae) Mastotermes pNPG Abs. NP NP Cazemier et al.,
darwiniensis 1997
(Rhinotermitidae) Coptotermes Salicin AC NP NP Mo et al., 2004
formosanus
Reticulitermes
flaviceps, R.
leptomandibularis
(Termitidae) Nasutitermes CB GOM NP NP Tokuda et al.,
takasagoensis 1997
N. exitiosus, CB GOP NP NP McEwen et al.,
N. walkeri 1980
Hymenoptera Scaptotrigona CB, pNPG, GOP NP NP Ferreira et al.,
(Apidae) bipunctata salicin, 1998
amygdalin
Lepidoptera Anticarsia pNPG, helicin, Abs., GOP, NP NP Yu, 1989
(Noctuidae) gemmatalis salicin, MUG, Flu
Heliothis zea CB
Spodoptera
frugiperda
Trichoplusia ni
S. frugiperda pNPG, MUG, Abs., Flu, SEC, 47 and Ferreira et al.,
CB, amygdalin, GOP AEC, 50 1998; Marana
gentiobiose, HIC et al., 2000
cellotriose
(Pyralidae) Diatraea pNPG, CB, GOP NP NP Ferreira et al.,
saccharalis salicin, 1998
amygdalin
(Sphingidae) Erinnyis ello pNPG, CB, GOP NP NP Ferreira et al.,
salicin, 1998
amygdalin
(Papilionidae) Parnassius apollo pNPG, CB DNSA, NP NP Nakonieczny
ssp. Abs. et al., 2006
frankenbergeri
(Saturniidae) Philosamia ricini CB AC NP NP Pant & Ramana,
1989
Orthoptera Abracis flavolineata pNPG, CB, Abs., GOP SEC, 82 Ferreira et al.,
(Acrididae) salicin, ABG, AEC 1998; Marana
lactose, LB et al., 1995
Locusta migratoria pNPG, CB Abs., GOP SEC 65 Morgan, 1975
Schistocerca pNPG Abs. NP NP Cazemier et al.,
gregaria 1997

Continued


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192 J. D. Willis et al.
Table 3 Continued
Order (family) Species Substrate
(Gryllidae) Acheata domesticus pNPG
Phasmatodea Eurycantha pNPG
(Phasmatidae) calcarata
Thysanura Thermobia CB, sucrose,
(Lepismatidae) domestica trehalose,
lactose
Ctenolepisma CB
lineata
Abbreviations: ABG, alkyl-β-glucosidase; Abs, absorbance; AEC, anion exchange chromatography; AC, alkaline copper; CB, cel-
lobiose; DGC, density gradient centrifugation; DC, differential centrifugation/precipitation; DNSA, dinitrosalicylic acid; Flu, fluo-
rescence; GOM, glucose-oxidase-mutarotase; GOP, glucose oxidase/peroxidase; HIC, hydrophobic interaction chromatography; LB,
laminaribiose; MUG, -methyl-umbelliferyl β-D-glucoside; NR, not reported; pNPG, p-nitrophenyl-β-D-glycosides; PE, preparative
electrophoresis; SEC, size exclusion chromatography; TB, tetrazolium blue.
termites, this approach has proved useful to sequence
endogenous and symbiont-produced EG, CBH and β-
glucosidases (Scharf et al., 2003; Todaka et al., 2007).
Both whole body (Lee et al., 2004; Wei et al., 2006b; Kim
et al., 2008) and midgut (Girard & Jouanin, 1999; Marana
et al., 2001) or salivary gland (Watanabe et al., 1998; Yuki
et al., 2008) cDNA libraries have been reported as useful
to identify endogenous insect cellulases. Due to limited
sequence information in some cases, these libraries are
usually used as sources to sequence expression sequence
tags (ESTs) that are then used for database searching to
identify putative cellulases (Girard & Jouanin, 1999; Lee
et al., 2004, 2005; Wei et al., 2006b; Kim et al., 2008;
Yuki et al., 2008). The advantage of this approach is that
multiple enzymes can be identified simultaneously, yet
their levels of activity cannot be assigned and may result
in identification of enzymes with low activity or with a
secondary role for cellulose digestion in the insect. Alter-
natively, partial sequence can be obtained from specific
cellulolytic proteins of interest and used to design probes
to screen cDNA libraries (Watanabe et al., 1998; Ferreira
et al., 2001; Marana et al., 2001) or primers for PCR am-
plification of full-length cellulase cDNAs (Li et al., 2003;
Sugimura et al., 2003). Availability of insect genomes
has facilitated the identification of endogenous cellulases
in Apis mellifera (Kunieda et al., 2006) and Tribolium
castaneum (Morris et al., 2009). Metagenomic projects
aimed at identifying cellulase genes from whole genome
shotgun libraries have proved successful in identifying
putative symbiont-derived cellulolytic enzymes in insects
(Todaka et al., 2007; Warnecke et al., 2007). Similarly,
Journal compilation 
C Institute of Zoology, Chinese Academy of Sciences, Insect Science, 17, 184–198
Detection Purifi- Size
Reference
method cation (kDa)
Abs. NP NP Cazemier et al.,
1997
Abs. NP NP Cazemier et al.,
1997
GOP NP NP Zinkler &
Gotze, 1987
AC NP NP Lasker & Giese,
1956
high throughput pyrosequencing projects have allowed
detection of multiple cellulase enzymes in Chrysomela
tremulae (Pauchet et al., 2009) and Melitaea cinxia (Vera
et al., 2008). The obvious advantage of these genomic
methods is the characterization of the complete insect cel-
lulolytic system, even though further research is necessary
to demonstrate functionality and specificity of the identi-
fied cellulases. With the increased availability of whole in-
sect genomes and next generation sequencing projects, the
number of identified endogenous and symbiont-derived
insect cellulases is expected to increase in the near future
(Matsui et al., 2009; Morrison et al., 2009).
A number of insect cellulolytic enzymes have been
expressed and purified in heterologous systems to
characterize their activity, specificity and stability
(Table 4). EG enzymes from Apriona germari (Lee
et al., 2004, 2005; Wei et al., 2006b) and Teleogryllus
emma (Kim et al., 2008) have been expressed as solu-
ble proteins and purified from Spodoptera SF9 cell cul-
tures. Even though the purified enzymes displayed the
expected β-1,4-endoglucanase activity, N-glycosylation
was reported to be necessary for activity of A. ger-
mari cellulases (Wei et al., 2005, 2006a). In comparison,
cellulases from Spodoptera frugiperda and Coptotermes
formosanus have been expressed and purified as ac-
tive enzymes in Escherichia coli (Marana et al., 2004;
Zhang et al., 2009a), suggesting that in this case gly-
cosylation may not be necessary for enzymatic activ-
ity. However, in the case of C. formosanus cellulase, it
was reported that C-terminal tagging, a process which
greatly facilitates recombinant enzyme purification,

C 2010 The Authors
 Methods for insect cellulases 193

Table 4 Insect-derived cellulases that have been cloned and heterologously expressed for their characterization. NP = not provided
by the authors.

Expression pH Thermo-
Order (family) Species Activity Reference
system optima stability

Coleoptera Apriona Sf9 cells β-1,4 endoglucanase 6.0 50–60◦ C Lee et al., 2005;
(Cerambycidae) germari Wei et al.,
2006b
Isoptera Neotermes E. coli β-glucosidase 5.0 45◦ C Ni et al., 2007b
(Kalotermitidae) koshunensis
(Rhinotermitidae) Coptotermes E. coli β-1,4 endoglucanase 5.0 42◦ C Zhou et al.,
formosanus 2007
Reticulitermes E. coli β-1,4 endoglucanase 6.9 50◦ C Ni et al., 2007b
speratus
(Termitidae) Nasutitermes E. coli β-1,4 endoglucanase 7.2 45◦ C Ni et al., 2007b
takasagoensis
Lepidoptera Spodoptera E. coli β-glucosidase NP NP Marana et al.,
(Noctuidae) frugiperda 2004
Orthoptera Teleogryllus Sf9 cells β-1,4 endoglucanase 5.0 45◦ C Kim et al., 2008
(Gryllidae) emmaa

affects activity and stability of the enzyme (Zhang et al.,
2009a). Random DNA shuffling between termite cellu-
lases has been used to increase expression levels as well
as thermostability in the mutated genes (Ni et al., 2005,
2007). Similarly, expression of cellulase genes from ter-
mite symbionts in Aspergillus oryzae has been suggested
to be optimized by codon optimization (Sasaguri et al.,
2008). An alternative heterologous expression system
based on infection of Bombyx mori larvae with transgenic
nucleopolyhedrovirus containing an insect cellulase gene
has also been reported to be efficient for insect cellulase
production (Lee et al., 2006). In this system, active cellu-
lase was recovered as a soluble protein in the hemolymph
of infected larvae.
Optimization of heterologous expression of insect cel-
lulases would be necessary to produce the high enzyme
amounts needed for lignocellulose degradation during
biofuel production. Alternative approaches to lignocellu-
lose degradation and preprocessing of feedstock, such as
inducible expression of cellulases in plants (Taylor et al.,
2008), have not been tested with insect enzymes.
Conclusions/insights from insect cellulases
and their applications to biofuels
Since diverse lignocellulosic feedstocks are being con-
sidered for biofuel production, optimized cellulase mix-
tures will be needed for each feedstock and pre-treatment
method used. Optimally, cellulolytic enzymes being used
in bioreactors for ethanol production would be stable un-
der high heat and acidic conditions used to make cellulose
in the lignocellulosic biomass available. High production
costs and activity limitations of currently available enzy-
matic mixtures highlight the need for cellulase prospect-
ing and improvement through genetic engineering (Lee,
1997; Wyman, 2007). Traditional biochemical and ge-
netic methods have demonstrated the presence of effec-
tive cellulolytic systems in insects, and have provided in-
sight on some of these enzymes. Broader understandings
of cellulose digestion in insects will continue to grow
under the advent of novel technologies. High through-
put metagenomic, transcriptomic and proteomic projects
should vastly increase our knowledge on the components
of these insect cellulolytic systems as well as their reg-
ulation. Expression of insect-derived cellulases in insect
cell cultures has proven successful to characterize speci-
ficity and activity of these enzymes, as well as to identify
key residues for activity (Marana et al., 2004). However,
and since glycosylation seems important for activity in
some cases (Wei et al., 2006a), further research would
be necessary to optimize expression of these enzymes
in bacterial or fungal systems to be used in bioreactors.
Similarly, expression of insect cellulases in plants (Oraby
et al., 2007) needs to be investigated as a possibility for
early pretreatment of lignocellulosic feedstocks.


C 2010 The Authors
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194 J. D. Willis et al.
Despite the low number of characterized insect cellu-
lases compared to fungal or bacterial counterparts, some
insect enzymes have been reported to display novel fea-
tures that may be of interest for biofuel production. For
example, an EG from Aulocophora foveicollis was re-
ported to display optimal activity at pH 7.8 (Sami &
Shakoori, 2008), which is unusual among animal (Watan-
abe & Tokuda, 2001) or even fungal (Lee, 1997) cel-
lulases. Genetic manipulation has also shown that insect-
derived cellulases are amenable to improvement for levels
of expression (Sasaguri et al., 2008), activity (Ni et al.,
2005), as well as thermostability (Ni et al., 2007). This
information highlights the promise of insect cellulolytic
systems to provide improvements to the production of
ethanol from plant biomass. Additionally, and consider-
ing the importance of these cellulases for insect survival
(Sami & Shakoori, 2008; Zhou et al., 2008), insect cellu-
lases may also be used as targets for the design of novel
insecticidal technologies.
Acknowledgments
JDW was supported by a grant from the Southeastern Sun
Grant Initiative Center
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Scharf, M.E. (2007) Correlation of cellulase gene expression
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Accepted December 21, 2009

C 2010 The Authors
 Insect Science (2010) 17, 199–219, DOI 10.1111/j.1744-7917.2010.01340.x

REVIEW

Molecular approaches to study the insect gut symbiotic

microbiota at the ‘omics’ age

Weibing Shi1,2 , Ryan Syrenne1 , Jian-Zhong Sun3 and Joshua S. Yuan1,2,3,4

1 2
Department of Plant Pathology and Microbiology and Institute for Plant Genomics and Biotechnology, Texas A&M University, Texas,
3 4
USA, Biofuels Institute, School of the Environment, Jiangsu University, Zhenjiang, Jiangsu Province, China, and Advanced Research
Institute for Sustainable Energy (ARISE), Texas A&M University, Texas, USA

Abstract Insect gut symbiotic microbiota play essential roles in the growth, development,
pathogenesis and environmental adaptation of host insects. The molecular and systems
level analysis of insect gut symbiotic microbial community will allow us to discover
novel biocatalysts for biomass deconstruction and to develop innovative strategies for
pest management. We hereby review the various molecular biology techniques as applied
to insect gut symbiont analysis. This review aims to serve as an informative resource
for experimental design and research strategy development in the field. We first discuss
various strategies for sample preparation and their pros and cons. The traditional molecular
techniques like DGGE, RFLP and FISH are covered with respect to how they are applied
to study the composition, diversity and dynamics of insect gut symbiotic microbiota. We
then focus on the various ‘omics’ techniques. The metagenome analysis together with the
recent advancements in next-generation sequencing will provide enormous sequencing
information, allowing in-depth microbial diversity analysis and modeling of pathways for
biological processes such as biomass degradation. The metagenome sequencing will also
enable the study of system dynamics and gene expression with metatranscriptome and
metaproteome methods. The integration of different ‘omics’ level data will allow us to
understand how insect gut works as a system to carry out its functions. The molecular
and systems-level understanding will also guide the reverse design of next-generation
biorefinery.
Key words DGGE, insect gut, metagenomics, metaproteomics, symbiotic microbiota,
systems biology

Introduction – Why study insect gut symbionts?
Insects are one of the most diverse groups of living or-
ganisms on earth (Chapman, 2006; Erwin, 1982). Due to
their diverse behaviors and feeding habits, almost no ter-
restrial food source can escape the consumption by one
or more insect species. Despite the diversity, the highly
interdependent and well-regulated symbiotic interactions
Correspondence: Joshua S. Yuan, Department of Plant Pathol-
ogy and Microbiology, Texas A&M University, College Station,
Texas, 77843, USA. Tel: 979 845 3016; email: syuan@tamu.edu
with micro-organisms seem to be an important common
property for different insect species (Breznak, 2004).
The definition and importance of symbiosis
Symbiosis often refers to the long-term and mutually
beneficial interactions among different species. Symbi-
otic microbes living inside the host species are referred
to as endosymbionts, and the symbiotic microbes living
upon or outside an insect’s body are often defined as ec-
tosymbionts (Breznak, 2004). Based on previous studies,
endosymbionts are prevalent in a variety of insect species
such as scarab beetles, cockroaches, termites and so on


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C Institute of Zoology, Chinese Academy of Sciences
200 W.B. Shi et al.
(Brune, 2003; Dasch et al., 1984; Kane & Pierce, 1994b;
Kaufman et al., 2000). Overall, it is estimated that a ma-
jority of members of the Insecta are involved in some
type of symbiosis (Moran, 2002; Moran, 2007; Moran
& Telang, 1998). Considering that Insecta is the largest
group of invertebrates, it is important to study symbiosis
in various insect species to understand the evolutionary
and ecological significance of the predominant phenom-
ena. In particular, we need to better understand what roles
the symbiotic microbiota plays in plant–insect interaction
in terms of host selection and co-evolution of host–insect
relationships. From an application perspective, the study
of insect symbionts will help to discover novel biocata-
lysts for biomass deconstruction and develop innovative
strategies for pest management.
Function of insect symbiotic microbiota
The herbivore insect gut microbiota has been well-
established for at least two aspects of the function: the
nutrient biosynthesis and the biomass deconstruction. The
nutritional function of the insect endosymbiotic microbes
have been well studied by feeding experiments with un-
balanced or poor diets lacking essential nutrients such as
amino acids and vitamins (Douglas, 1998). Some feed-
ing experiments demonstrated that the insect endosym-
biont can help to produce nutrients that do not exist in
the food (Khachane et al., 2007; Tamas et al., 2002;
Tamas et al., 2008; van Ham et al., 2003). The genome se-
quence of an obligate symbiont Wigglesworthia glossini-
dia revealed many genes for nutrient biosynthesis and
transport (Akman et al., 2002). The phenomena are typi-
cal for symbiotic microbes, which often dedicate part of
their genomes for the benefit of the hosts (Moran, 2001;
Ochman & Moran, 2001). A recent metagenome project
also revealed that the viruses affecting the symbionts of
the honeybee will lead to detrimental effects on honeybee
growth and development and could be a major cause for
CCD (colony collapse disease) (Cox-Foster et al., 2007).
The second well-characterized function for insect sym-
biotic microbiota is the biomass deconstruction and di-
gestion function. Both herbivore insects and symbiotic
microbes can secrete cellulytic enzymes for biomass de-
construction and hydrolysis (Ohkuma, 2003; Tokuda &
Watanabe, 2007; Warnecke et al., 2007; Sun & Zhou,
2009). It has been controversial about which plays a more
important role for biomass deconstruction, the symbionts
or insect host itself. Despite the controversy, the impor-
tance of symbiotic microbes for biomass deconstruction
has recently been established by various genome-level
studies. For example, symbiotic microbiota can help ter-
mites to deconstruct lignocellulosic biomass with high
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C Institute of Zoology, Chinese Academy of Sciences, Insect Science, 17, 199–219
efficiency (Ohkuma, 2003). The termite gut has actu-
ally been referred to as the smallest bioreactor in the
world (Brune, 1998). The recent sequencing of the sym-
biotic microbiota of the higher termite revealed many
glycosyl hydrolase enzymes with activities for degrading
cell wall components such as cellulose and hemicellulose
(Warnecke et al., 2007). In addition, the recent comple-
tion of the genome sequence of a prokaryotic symbiont of
cellulolytic protozoa Pseudotrichonympha grassi has also
unveiled its ability to fix nitrogen and to recycle putative
host nitrogen wastes for the biosynthesis of diverse amino
acids and cofactors (Hongoh et al., 2008b). The protozoa
contains up to 70% of the bacterial cells in the gut of
the termite Coptotermes formosanus and is an important
component of the termite gut symbiont.
Both nutrient production and biomass deconstruction
functions of the insect gut symbiotic microbiota can be ex-
ploited for biotechnology purposes. On one side, it might
be possible to develop various strategies for pest manage-
ment through the control of insect gut symbiotic microbes.
On the other side, the insect gut symbiotic microbiota can
be exploited for novel biocatalysts and microbe strain dis-
covery. Combined with functional validation, these new
biocatalysts and microbe strains could greatly improve the
design and efficiency of the next-generation biorefinery.
The thorough understanding of the insect gut as a natural
biocatalyst system with various molecular techniques will
also enable the reverse design of next-generation biore-
finery. Regardless of the goal of analysis, the first task for
analyzing insect gut microbiota is to prepare the samples
that well represent the microbe community in the insect
guts.
Sample preparation for insect gut symbiotic
microbial study
At the ‘omics’ age, DNA, RNA, protein and metabo-
lite samples can be prepared from insect gut symbionts.
We hereby focus on metagenomic DNA sample prepara-
tion and then briefly discuss the sample preparation for
metaproteomics.
Insect gut metagenomic DNA extraction
Metagenomics can be defined as the study of the
metagenome, the whole genetic material of the microbial
community existing in certain eco-environments (Sleator
et al., 2008). The ultimate goal of metagenomics is to
acquire a global view of the composition and function
of the microbial community (Guazzaroni et al., 2009).
The proper methods for DNA extraction remain keys

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Table 1 Commercial kits for metagenome DNA extraction and their application in insect gut systems.
Company Target product
MP Biomedicals FastDNA SPIN Kit for Soil
Sigma-Aldrich GenElute bacterial Genomic DNA Kit
QIAGEN Qiagen DNeasy Tissue Kit
QIAGEN QIAamp DNA Mini Kit
Promega WizardTM Genomic DNA Purification Kit
Mo Bio PowerSoilTM DNA Isolation Kit
Laboratories
to reaching a comprehensive and unbiased evaluation of
metagenomes of the community, particularly for the un-
culturable micro-organisms (Cowan et al., 2005). In order
to reach such a goal, there are three aspects to consider dur-
ing the sample preparation (Schmeisser et al., 2007). The
first aspect is the coverage. Metagenomic DNA should
cover as many microbial species as possible. The sec-
ond aspect is the integrity of the DNA sample. Shearing
should be avoided to obtain high molecular weight and
high quality metagenomic DNA. The third aspect is pu-
rity. The metagenomic DNA should be free of contami-
nants interfering with downstream DNA processing such
as enzyme digestion, polymerase chain reaction (PCR)
and vector ligation (Schmeisser et al., 2007).
Many of the insect gut microbial DNA isolation proto-
cols were derived from those for soil microbial community
analysis and the first paper on the extraction of DNA from
soil was published more than three decades ago (Torsvik,
1980). Two strategies have been popular for metagenomic
DNA isolation, and they are the cell recovery method and
the direct lysis method (Roose-Amsaleg et al., 2001). The
cell recovery method isolates intact organisms from the
gut content prior to cell lysis, and the cell isolation is
achieved either by repeated homogenization and differen-
tial centrifugation (Holben et al., 1988; Hopkins et al.,
1991) or by gradient centrifugation in media such as su-
crose, Nycodenz R
, PercollR
or metrizamide (Pillai et al.,
1991; Robe et al., 2003). Some commercial kits have re-
cently become available and these kits greatly simplified
many cultivation-independent analysis methods (Smalla,
2004). The commercial kits used for DNA extraction from
insect gut systems are shown in Table 1. For instance,
Schloss et al. (2006) used FastDNA SPIN kit for soil (MP
Biomedical, Solon, OH, US) to isolate the metagenomic
DNA from wood-boring beetle gut after the sonication
and centrifugation separation of bacterial cells from in-

C 2010 The Authors
Journal compilation 
C Institute of Zoology, Chinese Academy of Sciences, Insect Science, 17, 199–219
Molecular approaches for insect gut symbiont study 201
Application in insect
Website
gut symbiota
http://www.mpbio.com Zhang & Jackson, 2008
Dillon et al., 2008
Shinzato et al., 2005
http://www.sigmaaldrich.com/ Guan et al., 2007
http://www1.qiagen.com/ Fisher et al., 2007
http://www1.qiagen.com/ Hosokawa et al., 2006
http://www.promega.com/Default.asp Wei et al., 2006
http://www.mobio.com/index.php Pittman et al., 2008b
sect gut wall. The DNA isolation involves mechanical
lysis by bead beating followed by purification of DNA on
a silica matrix (Schloss et al., 2006). The same kit has also
been used widely for metagenomic DNA extraction from
the gut systems of grass grub (Zhang & Jackson, 2008),
feral locusts, grasshoppers (Dillon et al., 2008) and ter-
mites (Shinzato et al., 2005). Other commercial kits used
for insect gut symbiotic microbial metagenomic DNA iso-
lation includes the GenElute bacterial genomic DNA kit
(Sigma-Aldrich Corp., St. Louis, MO, US) (Guan et al.,
2007), Qiagen DNeasy Tissue kit (Fisher et al., 2007),
QIAamp DNA Mini Kit (Hosokawa et al., 2006),
WizardTM Genomic DNA Purification Kit from Promega
(Wei et al., 2006), and PowerSoilTM DNA isolation kit
(Pittman et al., 2008b). Despite the available commer-
cial kits, one has to realize that the metagenomic DNA
preparation protocol has to be optimized because most of
these kits are not designed for metagenomic DNA iso-
lation from insect gut (Broderick et al., 2004; Warnecke
et al., 2007). For example, we have recently modified
an indirect DNA extraction method for various insect gut
symbiont metagenomic DNA extractions (Shi et al., 2009,
unpublished data).
Besides the cell separation approaches, another ap-
proach is based on direct or in situ lysis of microbial
cells in the presence of the environmental matrix (e.g.,
soil, sediments or plant material), followed by the sepa-
ration of nucleic acids from matrix components and cell
debris (Ogram et al., 1987). The strategy generally yields
more DNA and is believed to provide a better represen-
tation of environmental biodiversity (More et al., 1994).
However, the largest disadvantage of direct lysis methods
is the co-recovery of contaminants like humic and fulvic
acids with environmental DNA, and these contaminants
are visible as a dark color in the DNA sample. The contam-
inants have been demonstrated to be inhibitors for DNA
202 W.B. Shi et al.
hybridization, digestion and PCR (polymerase chain reac-
tions) (Jackson et al., 1997; Miller et al., 1999; Tebbe &
Vahjen, 1993). The removal of co-extracted humic acids
is critical for the direct lysis method. Lilburn et al. (1999)
used direct lysis method for phylogenetic diversity study
of termite gut spirochaetes (Lilburn et al., 1999). Despite
the advantages of the direct lysis method, much fewer
studies used the method to study the insect gut symbiont,
probably because of the concerns over contamination of
the host DNA. Overall, cell recovery method has been
much more popular in the insect gut metagenomic anal-
ysis and various commercial kits and modified protocols
are available for the analysis. The cell recovery method
can also be modified to isolate RNA from the symbiotic
microbiota.
Protein for ‘omics’ analysis
Besides the metagenomics, metaproteomics are also
important perspectives for analyzing insect gut mi-
crobe communities. Metaproteome describes the pro-
teins expressed in the environmental samples and pro-
vides the real-time dynamics of the system (Handelsman
et al., 1998). Among the various proteomic techniques,
mass spectrometry (MS)-based shot-gun proteomics has
emerged as the primary method for the identification and
quantification of protein expression (Cravatt et al., 2007).
As for metagenome analysis, sample preparation is also
crucial for metaproteomics. The challenges come from
requirements from both the environmental samples and
the ESI (electrospray ionization) MS analysis. On one
side, ESI is highly sensitive to detergent and requires the
sample to be relatively pure. The extra purification step
is often involved for sample preparation for shot-gun pro-
teomics and the use of detergent like sodium dodecyl
sulfate (SDS) should be avoided. On the other side, the
sample preparation from insect guts needs to be compre-
hensive and contamination from the host tissue needs to
be avoided. Several protocols were developed based on
the previous metaproteomics analysis of environmental
samples. Ogunseitan developed and evaluated two meth-
ods for extracting proteins from water, sediments and
soil samples (Ogunseitan, 1993, 1997). One is the boil-
ing method, which recovered high concentrations of pro-
teins from waste water but not from soil and sediments.
The other one is the freeze–thaw method, which worked
better for soils and sediments (Ogunseitan, 1993, 1997).
After the pioneering work, different extracting methods
were developed for various purposes (Schulze et al., 2005;
Singleton et al., 2003). As compared to the environmen-
tal samples like soil and sediment, the insect gut samples
are normally very limited and need specific modification
Journal compilation 
C Institute of Zoology, Chinese Academy of Sciences, Insect Science, 17, 199–219
of the protocols for efficient and comprehensive extrac-
tion of proteome for LC-MS/MS (liquid chromatography-
mass spectrometry/mass spectrometry) analysis. In
addition, the extraction of total microbial protein and the
extraction of free proteins in the gut content will be differ-
ent. Warnecke and colleagues employed metaproteomics
approaches to study the free proteins extracted from wood-
feeding higher termite hindgut (Warnecke et al., 2007).
The sample preparation involves high-speed centrifuga-
tion of luminal contents in saline buffer to remove the
insoluble fraction. The soluble proteins were then dena-
tured, reduced, alkylated, and digested with trypsin for
the LC-MS/MS-based shot-gun proteomics analysis. The
analysis allowed measurement of soluble proteins in the
gut contents. However, analysis of total microbial protein
will have to follow a protocol similar to the cell recovery
metagenomic DNA extraction method, where the micro-
bial cells will be first separated and then total protein will
be extracted. We have recently developed such a protocol
for cattle rumen metaproteomics analysis, which can also
be used for insect gut analysis.
Traditional molecular techniques to investigate
insect gut microbiota
Traditional molecular techniques played an important role
in furthering our understanding of the composition and
function of insect gut symbionts. These techniques con-
tinue to provide solutions for insect gut microbial commu-
nity analysis at the ‘omics’ age. Over the past two decades,
the study of insect gut samples with molecular methods
has revealed a large discrepancy between the relatively
few culturable micro-organisms and the significant diver-
sity present in insect gut (Head et al., 1998; Pace, 1997).
Due to the limitation of cultivation-based methods, it was
expected that most of the diversity in insect gut micro-
biomes were still unknown (Stokes et al., 2001). In order
to study the diversity of insect gut microbial communities,
three major molecular approaches have been employed to
discover new genes and investigate the composition of gut
microbial communities. These three approaches include
gene targeting PCR, molecular fingerprinting techniques
such as DGGE (denaturing gradient gel electrophoresis),
and oligonucleotide probe-based hybridization techniques
such as FISH (fluorescent in situ hybridization) (Stokes
et al., 2001).
Gene targeting: gene-specific PCR
Gene targeting techniques employ gene-specific
primers to specifically amplify target genes, including

C 2010 The Authors

conserved 16S rRNA gene or a gene of specific functional
interest from the metagenomic DNA of insect gut sym-
bionts. This approach has been widely applied to insect
gut symbiotic microbiota analysis and has revealed sub-
stantial bacterial diversity and groups of unculturable mi-
crobes (Brauman et al., 2001; Paster et al., 1996; Spiteller
et al., 2000). Kane and Pierce (1994a) were among the
first to use PCR-based ribosomal DNA sequencing to
study insect gut microbial communities. Later on, Mckil-
lip and colleagues analyzed the composition of the mi-
crobiome in the midgut of Pandemis pyrusana Kearfott
by both PCR and culturing techniques (McKillip et al.,
1997). Lilburn and colleagues sequenced 98 clones of
near-full-length 16S rDNA genes of Spirochaetes in the
gut of termite species Reticulitermes flavipes. The re-
search revealed substantial phylogenetic diversity in the
termite gut (Lilburn et al., 1999). Phylogenetic analy-
sis of 16S rRNA genes recovered from the hindgut of
soil-feeding termites also revealed an enormous diversity
of bacteria in the different gut compartments (Schmitt-
Wagner et al., 2003b). Based on the PCR targeting of
16S rRNA, it has also been shown that most of the gut
microbial 16S rRNAs from termite Reticulitermes sper-
atus were unknown (Ohkuma & Kudo, 1996). Most of
the early 16S rRNA gene targeting analyses revealed a
significant number of unknown bacterial species at the
time.
Besides 16S rRNA, gene-specific PCR has also been
widely used to discover genes of interest and survey
metabolic pathways. This approach has been particu-
larly useful in cell wall degrading enzyme discovery for
bioenergy purposes. A number of cellulases belonging to
glycosyl hydrolase family 45 were cloned by gene target-
ing from the flagellates Koruga bonita and Deltotricho-
nympha nana, both of which were cultured from termite
gut (Li et al., 2003). In addition, Inoue and colleagues
identified a cellulase gene from lower termite hindgut us-
ing PCR with gene-specific primers and in situ hybridiza-
tion (Inoue et al., 2005).
In addition to gene-targeting PCR of DNA samples,
reverse transcriptase PCR (RT-PCR) from RNA has also
been employed to clone genes from environmental sam-
ples (Manefield et al., 2002). By combining the RT-PCR
with immune-blotting, Casu and colleagues identified a
major excretory/secretory protease from Lucilia cuprina
larvae (Casu et al., 1996). Noda and colleagues also am-
plified a nitrogen fixation gene from microbial RNA in
the gut of the termite Neotermes koshunensis by RT-PCR
(Noda et al., 1999). RT-PCR experiments also revealed
that five GHF9 EG (Glycosyl Hydrolase Family 9 En-
doglucanase) homologs were expressed in the salivary
glands and the midgut of termites (Nakashima et al.,

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Journal compilation 
C Institute of Zoology, Chinese Academy of Sciences, Insect Science, 17, 199–219
Molecular approaches for insect gut symbiont study 203
2002). Other examples employing the RT-PCR technique
for gene discovery in insect guts includes studies in Ancy-
lostoma caninum hookworms (Jones & Hotez, 2002), Cre-
ontiades dilutus (Colebatch et al., 2002), Protaetia brevi-
tarsis (Yoon et al., 2003), Aedes aegypti (Pootanakit et al.,
2003), Helicoverpa armigera (Chougule et al., 2005),
and Manduca sexta (Brinkmann et al., 2008; Hogenkamp
et al., 2005).
Even though gene-specific PCR was proven to be effec-
tive for gene discovery and microbial diversity analysis,
two major limitations have restricted the application of the
technique (Cowan et al., 2005). First, the gene-targeting
techniques depend on existing sequence information to
design primers for PCR amplification, which greatly lim-
ited the application of the technique. Second, normally
only partial sequence of the genes can be cloned. The
cloning of full-length genes will have to involve further
PCR-based chromosome walking (Cowan et al., 2005).
The available next-generation sequencing techniques and
the metagenomic strategies will certainly revolutionize
both gene discovery and biodiversity analysis for the in-
sect gut symbiotic microbiota. In addition to traditional
gene-targeting PCR-based techniques, PCR can also be
used for various molecular fingerprinting techniques to
study microbial diversity.
Molecular fingerprinting techniques
Besides the library-based gene targeting PCR, several
other PCR-based techniques have also been widely used
to study microbial diversity in various environmental sam-
ples. These molecular fingerprinting techniques include
denaturing or temperature gradient gel electrophoresis
(DGGE or TGGE) (Muyzer et al., 1993; Muyzer &
Smalla, 1998), restriction fragment length polymorphisms
(RFLP) (Liu et al., 1997; Osborn et al., 2000), single
strand conformation polymorphism (SSCP) (Lee et al.,
1996; Schwieger & Tebbe, 1998), and random amplified
polymorphic DNA (RAPD) (Kauppinen et al., 1999). For
microbial diversity analysis, these techniques are usually
used to analyze the sequence of 16s rRNA from different
microbial species, where both molecular fingerprints and
phylogenetic affiliation of microbial species can be gen-
erated (Smalla, 2004). These techniques have been proven
to be helpful in providing an overview of microbial diver-
sity in certain insect gut symbiotic microbiota. We hereby
review the previous application of these techniques in in-
sect gut microbial diversity analysis.
Among the different aforementioned genetic finger-
printing techniques, DGGE is perhaps the most com-
monly used. Recent application of the technique to study
insect gut microbial diversity has led to a much more
204 W.B. Shi et al.
comprehensive understanding of insect symbionts (da
Mota et al., 2005; Schabereiter-Gurtner et al., 2003;
Smalla et al., 2007; Webster et al., 2003). The DGGE
profiling of wasp larval Vespula germanica revealed a
diverse group of micro-organisms in the gut and in-
dicated that the wasp larva are not dependent on one
particular type of mutualist (Reeson et al., 2003). Be-
har and colleagues analyzed Mediterranean fruit fly gut
bacterial communities using both culture-dependent and
culture-independent approaches such as DGGE and re-
vealed that the family Enterobacteriaceae was the most
dominant species in the fruit fly gut (Behar et al., 2005).
Recently, DGGE was employed to explore microbial di-
versity in herbivore insects to study the potential mech-
anisms for biomass degradation. Enterobacterial repeti-
tive intergenic consensus PCR (ERIC-PCR) and DGGE
were combined to compare the diversity of lactic acid
bacteria communities in wood- and soil-feeding termites
(Bauer et al., 2000). The DGGE method was also used
to survey and screen for gut micro-organisms in wood-
feeding termites (Hayashi et al., 2007), soil-feeding ter-
mites, and their mounds (Fall et al., 2007). In addition
to termites, the symbiotic microbiota in the hindguts of
scarab beetle larvae were also explored with metage-
nomic approaches mainly based on DGGE (Pittman et al.,
2008b; Vasanthakumar et al., 2006). Moreover, Dillon
and colleagues surveyed microbial diversity from four
species of feral locusts and grasshoppers by DGGE ana-
lysis of bacterial 16S gene fragments and revealed that
Gammaproteobacteria from the family Enterobacteri-
aceae is the most predominant species in grasshopper
and locust guts (Dillon et al., 2008). Recently, we re-
vealed the diversity of gut bacteria from different insect
species by DGGE and found significant microbial diver-
sity differences among wood-feeding, grass-feeding and
leaf-feeding insects (Shi et al., 2009, unpublished data).
DGGE has also been used to study symbiotic microbiota
in a variety of insect species such as Dermolepida albo-
hirturn (Pittman et al., 2008a; Pittman et al., 2008b),
Gadus morhua L. (McIntosh et al., 2008), diamond-
back moth (Raymond et al., 2008), Anopheles gambiae
(Lindh et al., 2008), Hippoglossus hippoglossus L.
(Bjornsdottir et al., 2009), and Artemia franciscana
(Orozco-Medina et al., 2009).
Restriction fragment length polymorphism (RFLP)
analysis differentiates homologous DNA sequences based
on the distinct DNA fragment patterns resulting from the
sequence specificity toward restriction enzymes (Esumi
et al., 1982). In 1993, Harada and Ishikawa used RFLP
to analyze 16S rRNA from the group of prokaryote mi-
crobes in the gut of the pea aphid. The result suggested
that gut microbes have a close relationship with aphid
Journal compilation 
C Institute of Zoology, Chinese Academy of Sciences, Insect Science, 17, 199–219
intracellular symbionts (Harada & Ishikawa, 1993). De-
spite this analysis, the application of traditional RFLP in
microbial diversity studies is very limited due to the in-
herent technical limitations of the technology. Domingo
used RFLP of 16S rRNA to study cricket hindgut micro-
bial communities and suggested that community RFLP
methods did not have sufficient resolution or specificity
required to study the effect of diets on cricket hindgut
microbial community dynamics (Domingo, 1998). Due
to the limitations of traditional RFLP, terminal restriction
fragment length polymorphism (T-RFLP) has been em-
ployed to study microbial diversity in insect gut (Shinzato
et al., 2005). Different from RFLP, T-RFLP will sepa-
rate homologous DNA based on the length and sequence
of the end sequence generated from restriction enzyme
digestion of 16S rRNA, which makes it much more effi-
cient in revealing microbial diversity. T-RFLP was used
to analyze the bacterial 16S rRNA genes in the midguts
of individual European cockchafer (Melolontha melolon-
tha) larvae and revealed a simple but variable commu-
nity structure (Egert et al., 2005). In addition, T-RFLP
has been used for gut symbiotic microbial community
research of various termites such as soil-feeding termites
(Donovan et al., 2004; Friedrich et al., 2001; Kohler et al.,
2008; Schmitt-Wagner et al., 2003a), wood-feeding lower
termites (Miyata et al., 2007; Stingl & Brune, 2003), and
fungus-growing termites (Hongoh et al., 2006; Mackenzie
et al., 2007; Shinzato et al., 2007). These studies helped
to reveal the composition and dynamics of termite gut
microbial communities and led to some speculations on
how symbiotic microbes could contribute to biomass
degradation.
Another traditional molecular fingerprinting technique
is random amplified polymorphic DNA (RAPD). The
analysis is based on amplification of genomic DNA using
random primers. RAPD-PCR was carried out to compare
microbiota composition between different generations of
western flower thrips Frankliniella occidentalis and re-
vealed a surprising result that some bacteria in the thrips
can be passed from generation to generation for up to
50 generations (de Vries et al., 2001a, b). The discovery
highlighted that symbiotic microbiota can be indigenous
instead of exogenous from the food material (de Vries
et al., 2001a, b). The application of RAPD is also very
limited due to technical complexity and low reproducibil-
ity of the technique.
Single-strand conformation polymorphism (SSCP) is
a technique that uses electrophoresis to separate single-
strand DNA to differentiate the homologous sequences
(Yandell, 1991). SSCP was introduced to insect gut mi-
crobiota analysis very recently and has not been widely
used. Mohr and Tebbe used SSCP to study the diversity

C 2010 The Authors

and phylogenetic consistency of bacteria in the guts of
three bee species at the same oilseed rape field (Mohr
& Tebbe, 2006). In a recent study, PCR-SSCP, RT-PCR-
SSCP and stable isotope probing (SIP) were combined
to study partial bacterial 16S rRNA genes to survey the
diversity of metabolically active bacteria in the larval gut
of Manduca sexta (Brinkmann et al., 2008).
Even though these different molecular fingerprinting
techniques have revealed significant microbial diversity
in the guts of various insect species, all of them are rather
limited in providing comprehensive and detailed analy-
sis of microbial diversity. The techniques are particularly
limited if we want to survey the dynamics of microbial
communities during biomass deconstruction. The recently
developed metagenomics platforms are rapidly replacing
these molecular fingerprinting techniques.
Fluorescent in situ hybridization
Fluorescent in situ hybridization (FISH) is commonly
used in microbial ecology studies to visualize symbiotic
bacteria in the gut (Aminov et al., 2006; Cheung et al.,
1977). The application of FISH in insect gut microbial
studies often involves fluorescently labeled probes tar-
geting 16s rRNA with sequences specific for a bacterial
species or genus (Turroni et al., 2008). FISH has been used
to detect, visualize and characterize the intracellular sym-
biotic bacteria of aphids (Fukatsu et al., 1998), crickets
(Domingo et al., 1998), termites (Berchtold et al., 1999)
and some others. For biomass degradation-related stud-
ies, Berchtold and colleagues examined the abundance
and spatial distribution of major phylogenetic groups of
bacteria in the hindguts of the Australian lower termite
Mastotermes darwiniensis using FISH with group-
specific, fluorescently labeled, rRNA-targeted oligonu-
cleotide probes. The approach has been shown to be par-
ticularly useful in studying uncultivated microbes to ob-
serve the dynamics of microbiota (Santo Domingo et al.,
1998). However, when complex bacterial communities
from environmental samples are analyzed by FISH with
rRNA-targeted probes, several technical problems and po-
tential artifacts might occur and the detailed composition
of the microbiota cannot be revealed. In addition, bacteria
in less nutrient-rich environments have low ribosome con-
tent, which will affect the sensitivity of detection (Smalla,
2004). In complement to FISH, DAPI (4 ,6-diamidino-2-
phenylindole) and GFP (green fluorescent protein) have
also been used to visualize microbial communities. DAPI
staining of bacterial cells highlighted the significant dif-
ferences in the number of bacterial cells among differ-
ent insect species when reared under the same conditions
(Cazemier et al., 1997a, b). GFP can be used to track tar-

C 2010 The Authors
Journal compilation 
C Institute of Zoology, Chinese Academy of Sciences, Insect Science, 17, 199–219
Molecular approaches for insect gut symbiont study 205
get microbial species in the host. It has been used to show
that the colonization of bacterium Serratia entomophila in
the gut of the host Costelytra zealandica was not confined
to a specific site in the gut (Hurst & Jackson, 2002).
Overall, the various molecular techniques have greatly
advanced our understanding of insect gut microbial com-
munities, and many of these techniques will continue to
be important to further our understanding of insect gut
symbionts today. However, due to the inherent limita-
tions of these techniques, they cannot provide detailed
information regarding the gene and pathway for differ-
ent biological processes and a comprehensive coverage
of microbial taxonomy in the gut. In order to understand
the biological processes involved in biomass degradation,
we have to reach a detailed understanding of the biocata-
lysts, pathways and compositions of insect gut symbionts.
The recently available different ‘omics’ platforms enabled
such studies.
Techniques for “meta-omics” analysis of insect
gut symbionts
The recent advances in ‘omics’ technologies enabled
us to explore micro-organism communities in an un-
precedented way (Allen & Banfield, 2005; Tyson et al.,
2004). The high-throughput metagenome, metatranscrip-
tome and metaproteome analysis of micro-organism pop-
ulations will allow molecular, organism and population-
level investigation of how chemical and biological
processes have enabled, controlled and evolved (Allen
& Banfield, 2005). The complementary data annotation
and high-throughput functional screening will allow the
identification of novel catalysts and strains for bioreme-
diation, biomass processing, bioproduct synthesis and so
on (Hongoh et al., 2008a; Lorenz & Eck, 2005; Warnecke
et al., 2007). The so-called ‘metagenomics’ often in-
volves sequencing genomic DNA extracted from a mi-
crobe population in a certain eco-environmental setting
(Handelsman, 2004). It often involves sequence-based,
compositional and/or functional analyses of the com-
bined microbial genomes contained within an environ-
mental sample such as the insect gut (Handelsman et al.,
1998). Metatranscriptomics refers to sequencing analysis
of mRNA from a microbial population. Metaproteomics
refers to the quantification and identification of all the
proteins in a microbial community.
The different ‘meta-omics’ techniques have been
broadly used to explore the function and dynamics of di-
verse microbe populations in various eco-environmental
systems (Green et al., 2008; Keller & Zengler, 2004;
Strom, 2008). From the human intestine to the depths of
206 W.B. Shi et al.
the ocean, metagenomes from microbe communities have
been sequenced and analyzed for evolutionary, patholog-
ical, physiological, environmental and ecological studies
(Allen & Banfield, 2005; Tyson et al., 2004). The diver-
sity, composition and dynamics of a microbial community
largely defines its effectiveness, specificity and reactivity
for a certain function related to life, biogeochemical cycles
and environmental mitigation (Allen & Banfield, 2005;
Backhed et al., 2005; Falkowski et al., 2008; Green et al.,
2008; Keller & Zengler, 2004; Lorenz & Eck, 2005; Tyson
et al., 2004). In the past two decades, much effort has been
dedicated to exploring the components of microbial com-
munities from different niches at the molecular, organ-
ism and ecological level to discover novel enzymes, path-
ways and organisms for various applications (Green et al.,
2008; Roussel et al., 2008). For example, metagenome and
metatranscriptome sequencing have also become impor-
tant approaches for exploring biomass degrading mecha-
nisms in wood-feeding insects. Several studies have been
carried out to study symbionts in the hindgut and midgut
of wood-feeding higher termites (Warnecke et al., 2007)
and lower termites (Todaka et al., 2007; Hongoh et al.,
2008a, b). The termite is believed to recycle up to 30% of
the total carbon on earth, and the highly efficient ligno-
cellulosic biomass deconstruction has made the termite a
potential source for novel biocatalysts for biomass decon-
struction (Hongoh et al., 2008a; Warnecke et al., 2007).
Recent studies have indicated that symbiotic bacteria and
protozoa in the hindgut of the termite play an impor-
tant role in the hydrolysis of cellulose and hemicellu-
lose (Nakashima et al., 2002; Tokuda & Watanabe, 2007;
Warnecke & Hugenholtz, 2007; Warnecke et al., 2007;
Wheeler et al., 2007; Zhou et al., 2007). These analyses
not only revealed a diverse group of bacteria covering
12 phyla and 216 phylotypes, but also led to more than
100 candidate glycoside hydrolases. Moreover, the study
also indicated other important functions of symbiotic mi-
crobiota, including hydrogen metabolism, carbon dioxide-
reductive acetogenesis, and nitrogen fixation (Warnecke
et al., 2007). Overall, the development of metagenomics,
metatranscripomics and metaproteomics over the past
decades has been focused on the better understanding of
microbial diversity and function in the eco-environment,
and has been driven by increasing demands for biocat-
alysts and biomolecules for applications such as biore-
finery (Schmeisser et al., 2007). We hereby review the
application of these ‘omics’ platforms to study in-
sect gut symbiotic microbiota from several perspec-
tives, including the overview of metagenome analysis
of microbial communities, next-generation sequencing
and metagenome sequencing, functional metagenomics,
metatranscriptomics and metaproteomics.
Journal compilation 
C Institute of Zoology, Chinese Academy of Sciences, Insect Science, 17, 199–219
Metagenome sequencing and next-generation
sequencing
There are two principal metagenomic strategies for
metagenomics, the sequence-based metagenomics ap-
proach and functional metagenomics (Fig. 1). Sequence-
based metagenomics involves metagenome sequencing
and downstream data analysis. Functional metagenomics
involves screening of DNA or cDNA library for gene dis-
covery. Sequence-based analysis of metagenomic DNA
from insect gut symbionts has been well-established
during the past decade. Metagenomics was first car-
ried out with the conventional Sanger sequencing tech-
niques (Smalla, 2004). Sanger sequencing is more used
toward the 16s rRNA library or metagenomic DNA li-
brary (Smalla, 2004). The aforementioned metagenomic
analysis of termite hindgut symbiotic microbiota involves
Sanger sequencing of the metagenomic DNA library. To-
tal metagenomic DNA from pooled P3 luminal contents
was purified, cloned and sequenced (Warnecke et al.,
2007). Approximately 71 million base pairs of sequence
data were generated and assembled. The assembled se-
quences are highly fragmented. In order to better under-
stand the shot-gun data, 15 fosmids were selected for
further sequencing and training of the dataset. The data
have led to a comprehensive coverage and quantifica-
tion of the microbial composition in termite gut sym-
bionts. In addition, more than 700 glycoside hydrolase
(GH) catalytic domains corresponding to 45 different
CAZy families were identified through the analysis. The
study highlighted how metagenome sequencing can help
to identify natural biocatalysts, including different cellu-
lases and hemicellulases (Warnecke et al., 2007). Another
successful metagenome analysis is from the study of aphid
symbionts showing that heat tolerance of the host aphid
species can be conferred by gene mutation in their symbi-
otic microbes, which confers an evolutionary advantage
for the host in the field (Harmon et al., 2009).
The recent development of next-generation sequenc-
ing has offered the potential to revolutionize metagenome
analysis (Marusina, 2006). When next-generation se-
quencing is used, the approach can be the direct shot-
gun sequencing of metagenomic DNA. Up to now, four
major next-generation sequencing platforms have been
available. 454 sequencing technology is the first available
next generation sequencing technique and the platform is
based on ‘pyrosequencing’ and emulsion PCR amplifica-
tion (Margulies et al., 2005). The sequence read length for
454 sequencing can be up to 400 bases and the through-
put is relatively lower at 400 million bases per run. The
advantage of the 454 sequencing is the read length, which
makes it easier for the sequence assembly in de novo

C 2010 The Authors

Microbial samples
Genomic DNA Transcriptomic
cDNA
PCR-DGGE
Sequence-
based analysis
Solexa 454 GS20
GA platform pyrosequencing
Sequence assembly and
data analysis
ORF identification
Gene predictions
Metabolic modeling
Phylogenetic analysis
Reverse design of biorefinery, reconstitution
Fig. 1 ‘Omics’ analysis of insect gut as a natural biocatalyst system.
sequencing (Shendure & Ji, 2008; Yuan et al., 2008). Il-
lumina genome analyzer, formerly known as Solexa, is
based on the concept of ‘sequencing by syntheis’ (SBS)
(Adams et al., 2009; Mardis, 2008). With the latest de-
velopment of the technology, Illumina genome analyzer
can generate pairwise sequencing of 100 base pairs and
40 gigabase sequences per run. Another two platforms
are ABSOLiD and Helocus, both of which have simi-
lar sequencing throughput and less sequence read-length
(Mardis, 2008). For this reason, 454 and Illumina have
been the major approaches for metagenome sequenc-
ing. The advantage of 454 is the longer read length,
while the strength of Illumina is the sequence through-
put (Stangier, 2009). It is expected companies like Pa-
cific Biosciences will soon have the next-next-generation
sequencing techniques available. The accuracy and cov-
erage of the metagenome analysis highly depends on
the sequence coverage depth. The capacity of the next-
generation sequencing technique has enabled a deeper
coverage of the metagenomes and allows better annota-
tion of more genes.
Considering the pros and cons for Solexa and 454 se-
quencing technology, some recent studies have combined
the analysis with the two platforms to allow both better
assembly of the sequence, and the deeper coverage of the
genome (Ansorge, 2009; Shendure & Ji, 2008). Despite
the limitations of next-generation sequencing techniques,
they have been broadly used for metagenome sequenc-

C 2010 The Authors
Journal compilation 
C Institute of Zoology, Chinese Academy of Sciences, Insect Science, 17, 199–219
Molecular approaches for insect gut symbiont study 207
Insect guts Total proteins
Total chemical Enzyme assay
Compounds Enzyme digestion
NMR, MS, LC,
GC, analysis
Shotgun LC-MS/MS
Database search
and analysis
Modeling of
Discovery of novel coordinative
biocatalysts and function of
Microbial Species enzymes
of enzyme mixture
ing of environmental microbial communities from dif-
ferent niches, including soil (Blaha et al., 2007; Tringe
et al., 2005; Voget et al., 2003), the human gastrointesti-
nal tract (Gill et al., 2006), human feces (Breitbart et al.,
2003), the oceans (Culley et al., 2006; Venter et al., 2004),
the rumen (Brulc et al., 2009), acid-mine drains (Tyson
et al., 2004) and Zodletone Spring, OK, US (Elshahed
et al., 2005). However, more limited efforts have
been employed in insect gut symbionts. Very recently,
the next-generation-based metagenomic analysis of the
grasshopper (Orthoptera) and cutworm (Lepidoptera) gut
symbiotic microbiota were carried out to compare the
differences in community structure as related to feeding
habits and to discover novel genes for biomass degrada-
tion (W.B. Shi, X. Zhou, L.T. Liu, P. Gao, X.Y. Chen, N.
Kyprides, E.G. No, S.Y. Dai and J.S. Yuan, unpubl. data).
The analysis has led to the discovery of numerous novel
biocatalysts.
Functional metagenomics
Functional metagenomics involves screening for target
genes in a library built with metagenomic DNA or RNA
(Allen et al., 2009). Traditionally, metagenomic DNA can
be stored stably as a DNA library for further investigation.
In a similar way, RNA can be extracted to build a cDNA
library. The information held within a DNA or cDNA li-
brary can be used to determine community diversity and
208 W.B. Shi et al.
search for the enzymes with a particular activity (Steele
& Streit, 2005). For the DNA library, the basic steps of li-
brary construction include the extraction of metagenomic
DNA as aforementioned, the generation of suitably sized
DNA fragments, and the cloning of these fragments into
an appropriate vector (Cowan et al., 2005). For the cDNA
library, total RNA will be extracted and cDNA will be
synthesized for building into a proper vector. Both types
of libraries can be screened for genes of interest via DNA
hybridization using the probes of target genes or homolog
genes (Demaneche et al., 2009). The approach has been
used to search for various genes from insect guts. For
example, Shen and Jacobs Lorena reported the cloning
and characterization of a novel chitinase gene expressed
specifically in the midgut of adult Anopheles gambiae
females (Shen & Jacobs Lorena, 1997). They cloned the
chitinase gene from a cDNA library via screening and
further confirmed by Northern blot that the chitinase is
expressed exclusively in the guts of adult females.
One of the major limitations of the traditional screening
strategy is the need for probes specific to a certain gene.
The sensitivity and reproducibility often also depends on
the probe design. The combination of library screening
with gene expression and/or enzyme activity assay has
been developed to overcome such limitations. The method
has been successfully applied to discover new genes and
enzymes with different activities. A cDNA clone encod-
ing carboxypeptidase was isolated from a larval gut li-
brary of Helicoverpa armigera, and the complete cDNA
sequence was expressed in insect cells using the bac-
ulovirus system to verify carboxypetidase activity (Bown
et al., 1998). Girard and Jouanin isolated a cDNA encod-
ing chitinase of Pheadon cochleariae from a larval gut
library (Girard & Jouanin, 1999). For bioenergy research,
novel xylanases with distinct domains have been discov-
ered using metagenomic libraries of microbiota in several
insects belonging to Isoptera (termites) and Lepidoptera
(moths) (Brennan et al., 2004). Considering that this strat-
egy does not require the homolog sequences for genes
of interest, it has the potential to identify entirely new
classes of genes of new or known function (Handelsman,
2004). However, the heterologous gene expression also
has some limitations, including low gene expression level
and wrong post-translational modification (Handelsman
et al., 2002).
A recent development of functional metagenomics is
the use of biosensor technology in gene discovery from in-
sect symbioints. Guan and colleagues at the University of
Wisconsin constructed a metagenomic library consisting
of DNA extracted directly from gypsy moth midgut micro-
biota, and analyzed it using an intracellular screen desig-
nated as METREX (Guan et al., 2007). In this method, the
Journal compilation 
C Institute of Zoology, Chinese Academy of Sciences, Insect Science, 17, 199–219
biosensor detects compounds that induce the expression
of GFP from a bacterial quorum promoter by fluores-
cence microscopy or fluorescence-activated cell sorting
(Williamson et al., 2005). The authors identified an ac-
tive metagenomic clone encoding a mono-oxygenase ho-
mologue that mediates a pathway of indole oxidation. It
was the first to identify a new structural class of quorum-
sensing inducer from uncultured bacteria.
The functional metagenomics based on the cDNA li-
brary allows us to identify novel enzymes and genes for
a particular application; however, the analysis is limited
by the available probes for cDNA library screening and
the assay used for protein function determination (Chaves
et al., 2009; Moran et al., 2008). A more comprehensive
approach is to sequence the metatranscriptome of micro-
bial communities and annotate the metatranscriptome to
discover the novel genes.
Metatranscriptomics
Metatranscriptome involves the analysis of RNA in a
microbial community. RNA is converted to cDNA for the
analysis. The random sequencing of cDNA thus may lead
to a high percentage of rRNA signals. Different strategies
have been developed to remove rRNA to improve the cov-
erage of mRNA. In addition, the available next generation
sequencing technique has greatly enhanced the capacity
to carry out metatranscriptome analysis.
Cox-Foster and colleagues (Cox-Foster et al., 2007)
used an unbiased metatranscriptomic approach to char-
acterize microflora associated with honeybee Apis mel-
lifera in a search for the cause of colony collapse dis-
order (CCD). In this study, total RNA was extracted to
capture RNA viruses in presumed CCD-positive and neg-
ative bees for 454 sequencing. The raw sequencing reads
were trimmed and assembled into contigs, and then an-
alyzed using BLASTN and BLASTX for function anno-
tation. This analysis revealed the presence of bacteria,
fungi, parasites, metazoans and viruses in the bee gut
content. For example, sequences homologous to bacte-
rial 16S ribosomal RNA were assembled into 48 contigs.
Eighty-one distinct fungal 18S rRNA sequences were re-
covered from the pooled samples. More importantly, the
RNA profiling indicated that CCD may be caused by
the virus disruption of microbial community structure in
the bee gut system (Cox-Foster et al., 2007). More re-
cently, a parallel metatranscriptome analyses was used
to identify host and symbiont contributions in collabo-
rative lignocellulose digestion by termites (Tartar et al.,
2009). In this study, over 10 000 expressed sequence tags
(ESTs) were sequenced from host and symbiont libraries
that aligned into 6 555 putative transcripts, including 171

C 2010 The Authors

putative lignocelluase genes. They found that cellulases
were contributed by host plus symbiont genomes, whereas
hemicellulases were contributed exclusively by symbiont
genomes. However, ligninase, antioxidant and detoxifica-
tion enzymes were identified exclusively from the host
library.
These researches highlighted the importance of the in-
sect symbionts for host health and showed how the meta-
transcriptome can be applied to study insect gut systems.
The advantage for metatranscriptome sequencing is that it
can better reflect the dynamics and function of the insect
gut symbionts.
Metaprotoeomics techniques for insect gut symbiont
studies
Another way to explore systems dynamics is to study
the metaproteomics of insect gut symbionts. Like any
genome sequencing project, metagenome sequencing is
only the first step toward a comprehensive understanding
of composition, dynamics and function of insect gut sym-
biotic microbiota. The sequence itself won’t allow us to
understand the protein activity and the dynamic changes
of the system (Nelson, 2008). Post-genomic molecular ap-
proaches such as proteomics will allow us to study the ulti-
mate functional products of genes/genomes and derive the
function and dynamics of insect gut system. The collec-
tive study of all proteins in microbial communities, such
as those in insect gut, is referred as ‘metaproteomics’, to
distinguish from the proteomics study of single species
(Nelson, 2008). Metaproteomics allows the measurement
of gene expression from the perspective of presence and
abundance of translated proteins (Blackstock & Weir,
1999; Wilmes & Bond, 2004). The proteomics platform
can be generally classified as gel-free or gel-based sys-
tems (Kan et al., 2005). The traditional approach is to
analyze the protein sample with two-dimensional poly-
acrylamide gel electrophoresis (2D-PAGE) at first and
then further cut the spot for MS-based protein identi-
fication. The MS techniques that can be used for pro-
tein identification include both matrix-assisted laser des-
orption ionization (MALDI) and electrospray ionization
(ESI). MALDI is often coupled with time-of-flight (TOF)
mass analyzer, while ESI can be coupled with a vari-
ety of mass analyzers. The earliest approach for protein
identification of gel spot is through peptide fingerprint-
ing, where the peptides from protease-digested protein
will be measured by MALDI-TOF for the m/z value. The
pattern of peptide distribution will be searched against
a database of candidate proteins for identification. Even
though the method was successfully applied for protein
identification in gel-based proteomics, the accuracy and

C 2010 The Authors
Journal compilation 
C Institute of Zoology, Chinese Academy of Sciences, Insect Science, 17, 199–219
Molecular approaches for insect gut symbiont study 209
reproducibility of the method is often inconsistent. In par-
ticular, the post-translational modification of the protein
will severely distort the m/z value for the protein identi-
fication. For this reason, peptide fingerprinting has been
gradually replaced with tandem MS (MS/MS) analysis,
where individual peptides will be subject to two rounds
of MS analyses. The first round of MS analysis will ren-
der the m/z value of the peptide, and the peptide will be
further broken into fragment ions by electron or chem-
ical dissociation for the second round of measurement.
According to the fragment ion pattern, a protein sequence
can be identified based on the search for fragment patterns
against the database with protein sequences. The tandem
MS method has become the most popular approach for
protein identification.
Even though gel-based proteomics was the golden stan-
dard for proteomics, the 2D-gel-based methods have nu-
merous inherent limitations including low sensitivity, low
coverage of proteome and difficulties in quantification.
For all these reasons, gel-based proteomics has been
gradually replaced with the gel-free proteomics, which
mainly relies on LC-MS/MS platform. The most pop-
ular approach for gel-free proteomics is MudPIT (mul-
tidimensional protein identification technology)-based
shot-gun proteomics (Delahunty & Yates, 2007; Lohrig
& Wolters, 2009). In this approach, the total pro-
tein from a sample is first digested by protease into
a peptide mixture and the peptide mixture is further
separated by multidimensional LC. The separated pep-
tides are further analyzed by MS/MS for protein iden-
tification as aforementioned. MudPIT can be com-
bined with the different labeling techniques like ICAT
(isotope coded affinity tags), ICPL (isotope coded protein
labels), or iTRAQ (isobaric tag for relative and absolute
quantification) for protein quantification (Delahunty &
Yates, 2007). MudPIT can also be used as a label-free
platform, where peptide quantification can be based on
total ion counts and numbers of peptides (Delahunty &
Yates, 2007). Despite the broad application of proteomics
techniques in various studies, the use of proteomics in the
analysis of insect gut symbiotic microbiota is still very
limited. In the aforementioned termite gut metagenomics
analysis, the authors carried out a proteomics analysis of
total gut protein to examine which enzymes are expressed
(Warnecke et al., 2007). The total proteins were first ex-
tracted from P3 luminal contents of wood-feeding higher
termites as aforementioned. The digested peptides were
then subject to three-dimensional LC-MS/MS analysis
for protein expression analysis. The fragment ion patterns
from metaproteomics were searched against a sequence
database derived from metagenome sequencing for pro-
tein identification. The study has revealed that expression
210 W.B. Shi et al.
of glycosyl hydrolases are regulated at the protein level,
and enzymes in the metagenome were not expressed at the
same time and same level (Warnecke et al., 2007). Further
study of the metaproteome in the natural biocatalyst sys-
tems such as termite gut will allow us to understand how
enzymes coordinate to degrade plant cell walls. Metapro-
teomics analysis will be based on the metagenome se-
quencing data and will help to further understanding of
insect gut symbiotic microbes to the proteome level.
Looking into the future
The study of insect symbiotic microbiota is important for
insect physiology, pest management, evolutionary study
and discovery of various biocatalysts for different applica-
tions, including pest management and biorefinery devel-
opment. In particular, the gut systems of many herbivore
insects can be considered as effective bioreactors, where
biomass material can be deconstructed for the synthesis
of various bioproducts important for insect growth and
development (Breznak, 2004). The coordinative function
of host and symbiont enzymes plays important roles in
biomass processing and degradation. The study of insect
gut symbiotic microbiota at the systems level will enable
us to reverse-design the next-generation biorefinery.
The techniques to study insect gut symbionts have ex-
perienced dramatic changes during the past two decades.
The initial studies of insect gut symbionts were based on
microbial culture-dependent platforms, which provided
very limited information for the diversity and functions
of insect gut symbiotic microbiota (Amann et al., 1995;
Dillon & Dillon, 2004). The culture-dependent technique
only allows us to access to a small portion of the
microbe community in insect guts (Oliver, 2000). The
culture-dependent analysis was quickly replaced and
complemented by molecular biotechniques independent
of microbial culturing. Methods like DGGE, SSCP, RFLP
and FISH allowed us to better explore the complexity
of natural microbial communities. These techniques
provided some speculations of microbial community
composition, dynamics and function. However, tra-
ditional molecular techniques still cannot provide a
comprehensive view of the composition and dynamics
of insect symbiotic microbial communities. The recently
developed metagenome sequencing techniques enabled
us to reach much deeper sequencing and better coverage
of the metagenome (Mardis, 2008). In particular, the
advancements in next-generation sequencing techniques
allowed us to explore the metagenomes from insect gut
symbiotic microbiota to an unprecedented depth and com-
prehensiveness (Adams et al., 2009; Stangier, 2009). In
Journal compilation 
C Institute of Zoology, Chinese Academy of Sciences, Insect Science, 17, 199–219
addition, functional analysis, metatranscriptomics,
metaproteomics and metabolite profiling are all provid-
ing important information regarding the function of insect
hosts and symbionts from different perspectives. The
integration of information will lead to a systems-level
understanding of insect gut as the system for biomass
deconstruction, nutrient biosynthesis and so on. Despite
significant progresses, several aspects of research need
to be emphasized to better exploit insect gut systems for
various biotechnology applications.
First, more insect gut systems need to be studied with
various ‘omics’ techniques. Current research mainly fo-
cuses on the termite gut as the model system for biomass
degradation. Comprehensive metagenomics and meta-
trascriptomics were carried out to study termite gut sys-
tems (Tartar et al., 2009; Warnecke et al., 2007). However,
there are many other insect species with strong capacities
to degrade lignocellulosic biomass (Sun & Zhou, 2009).
The cellullolytic enzyme activity in grasshopper gut is
actually comparable to that of the termite gut (Shi et al.,
2010). The comparative analyses of different insect gut
systems will allow us to identify common and unique fea-
tures for degrading different lignocellulosic biomasses in
various insect gut systems. Such studies will also help to
understand the co-evolution of insect hosts and symbionts
toward different food sources.
Second, bioinformatics challenges for the assembly of
next-generation sequencing data need to be better ad-
dressed. Despite the potential of next-generation sequenc-
ing in increasing the sequencing coverage of metagenome,
sequence assembly for metagenome is much more chal-
lenging than single species, in particular for complex
systems. The more microbe species in a community,
the more complexity and overall genome size there will
be for insect gut symbiotic microbiota. Illumina genome
analyzer has the most potential for increasing sequence
coverage due to higher sequencing throughput and lower
per base cost. However, short sequence read length to-
gether with large overall genome size from this technology
make it extremely challenging to assemble metagenome
sequences. The recent development of several assemblers
for short sequences like SSAKE, VEVELT, ABySS and
Euler have provided solutions for the assembly of short
sequence reads of genome sequencing (Scheibye-Alsing
et al., 2009). However, the conditions used for single
genome assembly are not suitable for metagenome se-
quencing. On one side, we need to find the optimized
parameters and criteria for the assembly of metagenomes;
one the other side, these software packages need to be
further improved for metagenome sequencing.
Third, lignocellulose digestion models of insects con-
sider both host and symbiont. In particular, enzymes

C 2010 The Authors

secreted by host insect species play particularly impor-
tant roles in lignin degradation (Tartar et al., 2009). In
previous studies, the metabolism of monoaromatic model
compounds by termites and their gut microflora were stud-
ied; the results indicated that microbial degradation of
plant aromatic compounds can occur in termite guts and
may contribute to the carbon and energy requirement of
the host (Brune et al., 1995). The recent metagenome
and metatrascriptome sequencing of gut symbionts for
termite, grasshopper and cutworm has led to the finding
of very few lignin-degrading laccases, peroxidases or es-
terases (Tartar et al., 2009; W.B. Shi, X. Zhou, L.T. Liu,
P. Gao, X.Y. Chen, N. Kyprides, E.G. No, S.Y. Dai and
J.S. Yuan, unpubl. data). Metaproteomics will provide a
powerful solution toward the observation of how biocata-
lysts from the host and microbes work together to degrade
biomass. However, more sequencing information needs to
be available to enable such analysis. The study of coordi-
native function of host and symbiotic microbial biocata-
lysts will help to guide the reverse-design of biorefineries
and the reconstitution of effective enzyme mixtures for
biomass degradation.
Fourth, the integration of different ‘omics’ data into
systems-level understanding of insect guts will be impor-
tant for the reverse-design of artificial reactors mimicking
natural biocatalyst systems. Systems biology enables the
observation of biological systems and processes at an in-
tegrated view (Rachlin et al., 2006). The interaction, dy-
namics and network of multiple components in a system
will be modeled based on genome, proteome, metabolome
and transcriptome analyses (Rachlin et al., 2006; Vieites
et al., 2009). The accumulation of different ‘omics’ data
regarding insect gut systems will allow us to investi-
gate how different components and biocatalysts work
together to fulfill various functions, including biomass
degradation.
Overall, we are at a golden age of addressing basic
and applied questions involved in insect gut systems. In
particular, the recently available ‘omics’ techniques will
revolutionize the field with enormous data to enable un-
precedented understanding of insect gut symbiotic micro-
biota and their interactions with hosts. The systems-level
integration of this tremendous information will enable in-
depth understanding of natural biocatalyst systems, like
insect guts, toward providing novel solutions for next-
generation biorefineries.
Acknowledgments
This research was supported by the Texas Agrilife Re-
search Bioenergy Research Initiative, Sungrant and a spe-

C 2010 The Authors
Journal compilation 
C Institute of Zoology, Chinese Academy of Sciences, Insect Science, 17, 199–219
Molecular approaches for insect gut symbiont study 211
cial research initiative for Bioenergy sponsored by Jiangsu
University, China.
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Accepted February 22, 2010
 Insect Science (2010) 17, 220–236, DOI 10.1111/j.1744-7917.2009.01310.x

REVIEW

Arthropods and biofuel production systems in North America

Douglas A. Landis and Benjamin P. Werling

Department of Entomology, and Great Lakes Bioenergy Research Center, 204 Center for Integrated Plant Systems, Michigan State
University, East Lansing, Michigan, USA

Abstract Biomass harvest may eventually be conducted on over 100 000 000 ha of US
crop and forest lands to meet federally-mandated targets for renewable biofuels. Such large-
scale land use changes could profoundly impact working landscapes and the arthropod
communities that inhabit them. We review the literature on dedicated biofuel crops and
biomass harvest from forests to look for commonalities in arthropod community responses.
With expanded biofuel production, existing arthropod pests of biofuel crops will likely
become more important and new pests will emerge. Beneficial arthropods will also be
influenced by biofuel crop habitats, potentially altering the distribution of pollination and
pest control services to the surrounding landscape. Production of biofuel crops including
initial crop selection, genetic improvement, agronomic practices, and harvest regimes will
also influence arthropod communities. In turn, arthropods will impact the productivity and
species composition of biomass production systems. Some of these processes have the
potential to cause landscape-level changes in arthropod community dynamics and insect-
vectored plant diseases. Finally, changes in arthropod populations and their spatiotemporal
distribution in the landscape will have impacts on consumers of insects at higher trophic
levels, potentially influencing their population and community dynamics and producing
feedbacks to arthropod communities. Given that dedicated biofuel crops and intensified
biomass harvest from forests are still relatively uncommon in North America, as they
increase, we anticipate ‘predictably unpredictable’ shifts in arthropod communities and
the ecosystem services and functions they support. We suggest that research on arthropod
dynamics within biofuel crops, their spillover into adjacent habitats, and implications for
the sustainability of working landscapes are critical topics for both basic and applied
investigations.
Key words biodiversity, land use change, pest management

Introduction tion systems. Arthropods are key mediators of ecosys-

Recent interest in production of energy from plant
biomass has spurred global efforts to develop dedicated
biofuel crops and intensify biomass harvest from for-
est ecosystems. For simplicity, we hereafter refer to
both sources of biomass collectively as biofuel produc-
Correspondence: Douglas A. Landis, Department of Ento-
mology, 204 Center for Integrated Plant Systems, Michigan State
University, East Lansing, MI 48824-1311, USA. Tel: (517) 353
1829; fax: (517) 353 5598; email: landisd@msu.edu
tem function in terrestrial ecosystems and as such,
any changes in the way that humans appropriate plant
biomass for biofuel production has immense implica-
tions. At one level, herbivorous arthropods will act as
pests of biofuel crops, potentially reducing the quan-
tity or quality of biomass harvested. Improved man-
agement systems will clearly be needed to mitigate the
negative impacts of arthropods as plant pests. Alterna-
tively, in their roles as decomposers, pollinators, preda-
tors and parasitoids, arthropods will be beneficial to
biomass production systems. Moreover, beneficial arthro-
pods may obtain resources in biofuels that increase their


C 2010 The Authors 220
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C Institute of Zoology, Chinese Academy of Sciences

abundance and provision of services in the broader land-
scape. Increased adoption of biofuel crops may thus
change the ways arthropod-mediated ecosystem services
(Isaacs et al., 2009) such as pollination and pest suppres-
sion are distributed in agricultural landscapes. Finally,
because arthropods provide food for other organisms
(e.g., birds and mammals), any changes in arthro-
pod community structure will have indirect effects that
are certain to radiate throughout terrestrial food webs
(Polis et al., 1997). However, it is also certain that such
indirect effects are likely to be highly variable, with some
being beneficial, some neutral, and others detrimental
to ecosystem function. As society considers increased
biofuel production systems, entomologists have a unique
opportunity to examine their varied implications.
Current and future biofuel cropping systems
in North America
At present, the US produces about nine billion gallons
of ethanol annually (Anonymous, 2009b), primar-
ily from the fermentation of corn grain. In addi-
tion, approximately 700 000 000 gallons of biodiesel
(Anonymous, 2009a) are produced from soybean, canola
and other vegetable oils. These first-generation biofuels
are based on well-known technologies that use the edi-
ble portion of food crops. As such, they have been crit-
icized for contributing to increased food prices (Naylor
et al., 2007; Mitchell, 2008, but see also Trostle, 2008).
Second-generation biofuels are produced from lignocel-
lulose obtained from the inedible portions of food crops
(e.g., wheat straw and corn stover) or a non-food plant
(Schubert, 2006). Such cellulosic biofuels can be pro-
duced from biomass harvested form forests or dedicated
woody energy crops such as willow and poplar, as well
as from herbaceous plants such as switchgrass, miscant-
hus, and mixed prairie grasses and forbs (Perlack et al.,
2005). The resulting biomass may be directly burned to
generate electricity or co-fired with fossil fuels such as
coal. Alternatively, cellulosic biomass can be processed
via either thermochemical (i.e., pyrolysis) or enzymatic
platforms to produce a variety of liquid fuels and other
products (Ragauskas et al., 2006). The US Energy In-
dependence and Security Act of 2007 calls for an in-
crease in renewable fuel production from 8 billion gallons
in 2008 to 36 billion gallons in 2022. Cellulosic biofu-
els are to account for 16 billion gallons of this increase
(Anonymous, 2009b). Meeting these targets is an-
ticipated to require harvesting biomass from over
100 000 000 ha of US land (Graham et al., 2007;
Perlack et al., 2005; Schmer et al., 2008).

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C Institute of Zoology, Chinese Academy of Sciences, Insect Science, 17, 220–236
Arthropods and biofuel crops 221
In this review we explore what is known about the role
of arthropods in biofuel cropping systems. Our goal is
to provide an entry into the relevant literature. In ex-
ploring this literature, we highlight the major phenom-
ena (Table 1) that have emerged with increased pro-
duction of existing biofuel crops and draw on research
that provides lessons for future biofuel production sys-
tems (Table 2). In doing so, we also suggest key ar-
eas for future research. Our specific focus is on cur-
rent and proposed biofuel production systems in North
America. However, we draw on research from other re-
gions when it can inform the future development of biofu-
els in North America. Although specific biofuel crops will
differ throughout the world, the processes and concepts we
explore here should prove widely applicable. For example,
data from existing biofuel cropping systems from northern
Europe provide a wealth of information that can be applied
to North America. We also move beyond the borders of
biofuel crops to discuss the landscape-level implications
of increased biofuel production for arthropod communi-
ties and the ecosystem services that they provide (Losey
& Vaughan, 2006). It is important to note that we do not
focus on the use of arthropods for improved biomass pro-
cessing, as this is the focus of other contributions in this
special issue. In addition, for many of the traditional food
crops, pest management aspects have been previously re-
viewed. In these cases we simply refer to the relevant
reviews and focus on more recent literature and on novel
aspects encountered when the crop is used for biofuel
production.
Arthropods and herbaceous biofuel crops
Use of existing food crops for biofuel production
A number of existing crops are already used, or
could be used, for biofuel production in North America.
These include soybean (Glycine max (L.) Merr.) and the
oilseed Brassicas (B. rapa L. (campestris), B. juncea (L.)
Czern., and B. napus L.) for biodiesel production and
corn (Zea mays L.), sweet sorghum (Sorghum bicolor
(L.) Moench), sugar beet (Beta vulgaris L.) and sug-
arcane (Saccharum spp.) for ethanol production. Much
of the relevant pest management literature for these
crops has been previously reviewed: soybean (Kogan &
Turnipseed, 1987; Turnipseed & Kogan, 1976), oilseed
brassicas (Lamb, 1989), sorghum (Young & Teetes,
1977), sugar beet (Lange, 1987), sugar cane (Long
& Hensley, 1972) and corn (Brindley et al., 1975;
Chiang, 1978; Levine & Oloumi-Sadeghi, 1991; Gray
et al., 2009).
222 D. A. Landis & B. P. Werling
Table 1 Examples of the major ways in which arthropods are known to interact with biofuel production systems. References indicate
selected examples from the text.
Arthropod/biofuel crop interaction
Arthropods as pests of biofuel crops
Increased importance of existing pests
Emergence of new pests
Arthropods cause shifts in biofuel crop community composition or productivity
Biofuel crops alter arthropod pest dynamics in surrounding landscape
Potential to increase insecticide resistance
Potential for pest build-up and spillover into surrounding crops
Biofuel crops act as reservoirs for insect-transmitted diseases
Impacts on beneficial arthropods
Biofuel crop provides resources (shelter, food etc.) for beneficials
Biofuel crops alter spatial or temporal distribution of beneficials
Biofuel crops impact pollination/pest control services to surrounding
landscape
Biomass harvest patterns impact arthropod habitat
Community shifts
Food web shifts
Alter habitat for rare species
Change physical structure of habitat
Biofuel crop influences other trophic levels via arthropod subsidies
Bird nesting success
Pest complexes attacking widely-used crops can change
over time and space, suggesting that new pests will
continue to challenge entomologists in established pest
management systems. For example, the recent introduc-
tion of sweet sorghum into Greece for experimental bio-
fuel production revealed heavy infestations of the stalk
borer, Sesamia nonagrioides Lefebvre (Dimou et al.,
2007). This was the first report of a stalk-boring in-
sect in sorghum in this country. Similarly, Ward et al.
(2007) studied canola insect pests in Alabama in anticipa-
tion of increased production for biodiesel. They reported
the first known occurrence of the North American en-
demic clover stem weevil, Languria mozardi Latreille, on
canola. These examples suggest that host shifts onto bio-
fuel crops by existing insect fauna are likely to be an on-
going process, creating new pest management challenges
as production of existing crops expands to new areas.
Existing crops might also expand in acreage as they
are increasingly used for biofuels, potentially increasing
the availability of pest habitat and exacerbating control
problems. Specifically, demand for biofuels that are also
produced for food could pressure farmers to reduce ro-
tation intervals, double crop within years, or even move
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C Institute of Zoology, Chinese Academy of Sciences, Insect Science, 17, 220–236
Selected example(s)
Coyle, 2002; Hansen, 2003; Mattson et al., 2001
Dimou et al., 2007; Ward et al., 2007
Schmitz, 2008
Hansen, 2003
Ahmad et al., 1984; Semere & Slater, 2007
Huggett et al., 1999
Carmona et al., 1999; Menalled et al., 2001;
Gardiner et al., 2010
Frank et al., 2008
Reddersen, 2001; Landis et al., 2008
Nitterus & Gunnarsson, 2006; Ulyshen & Hanula,
2009
Hedgren, 2007
Jonsell et al., 2007
Kortello & Ham, 2010
Sage & Tucker, 1997, 1998
to continuous cultivation. In Denmark, oilseed rape pro-
duction has expanded and is anticipated to grow further
as demand for biodiesel increases. This has led to pro-
duction of winter- and spring-sown crops, extending the
period of bud availability for the pollen beetle, Meligethes
aeneus F. and forcing increased insecticide use. As a re-
sult, M. aeneus is now highly resistant to pyrethroids and
partially resistant to dimethoate, the two main groups of
insecticides used to control it (Hansen, 2003). Similarly,
increases in the extent of canola production in Australia
over time are correlated with increased densities of di-
amondback moth, Plutella xylostella (L.) a key pest of
brassicas (Schellhorn et al., 2008). Although not strictly a
result of biofuel crop production, these examples illustrate
the type of land use pressures that are likely to impact in-
sect management practices in the future. More generally,
expanding production of existing crops can profoundly
influence pest problems if these crops are more suitable
for pests than the crops they replace or if they provide
resources at key points in the pest’s life cycle (Kennedy
& Storer, 2000).
The expansion of biofuel crops might also indirectly
influence pest problems by affecting natural enemy

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 Arthropods and biofuel crops 223

Table 2 Ecological lessons and critical research avenues derived from examination of existing studies on arthropods in biofuel
production systems.

1. New pests of biofuel crops will continue to emerge. Endemic herbivores could include biofuels in their host range as production
expands to new areas.
2. Changes in the extent of biofuel production in existing production areas could alter the availability of habitat for existing pests,
exacerbating pest problems.
3. Expansion of biofuel production could negatively affect biocontrol if biofuel crops are unsuitable for natural enemies or replace
habitats that are critical for their persistence. Alternatively, some biofuel crops may be more suitable for natural enemies than
the crops they replace. These could provide habitat for natural enemies that contribute to control of pests, both in biofuel crops
and the broader landscape.
4. Existing data gives hints about the taxa that will become pests of biofuel crops. However, data should be collected across wider
geographic areas and the impact of pests on biomass yield should be quantified.
5. Biofuel crops show genetic variation in insect resistance. This variation could be mined to produce resistant cultivars.
6. Direct pests are not the only problem. Biofuels could also act as perennial reservoirs of viruses that can be transmitted to other
crops via mobile insect vectors.
7. It will be crucial to design and execute experiments that compare arthropod communities between candidate biofuel crops.
Existing data largely come from experiments that are not designed for this purpose.
8. The conservation benefits of biomass crops to arthropods and the organisms that use them as food have only received preliminary
attention.
9. Conservation benefits will not be uniform, but will be impacted by landscape context, within-site management and plant diversity.
10. The final impact of herbivores on biofuel productivity will depend on the full suite of interactions that occur between pests and
other members of the arthropod community. Abstracting these interactions from their community-context could lead to
misleading predictions about pests and biofuels.

populations. In the US, increased demand for corn as a
biofuel feedstock has led to increases in corn production
with negative impacts on biocontrol services in surround-
ing crops. Landis et al. (2008) reported that biocontrol of
the invasive soybean aphid decreased in soybean grown
in areas with increased corn acreage, costing growers a
minimum of US$58 million per year in lost yield and
increased pest management costs. This could occur be-
cause corn is unsuitable for natural enemies, or because
increases in corn production result in the loss of other key
habitats. For example, non-crop habitats (e.g., forests and
grasslands) appear to provide critical habitat for natural
enemies (Bianchi et al., 2006), and cultivating these habi-
tats to increase crop production could reduce biological
control.
Switchgrass
The US Department of Energy has been developing
switchgrass, Panicum virgatum L. as a biomass crop
since the early 1990s (McLaughlin & Kszos, 2005).
Literature on switchgrass production provides an excel-
lent example of the type of arthropod pest data that is,
and is not, available for relatively novel biofuel crops.
Existing data suggests that pests can affect the estab-
lishment of switchgrass, but may cause only minor dam-
age once established (Wolf & Fiske, 1995). Parrish et al.
(1999) reported that in Virginia newly emerged switch-
grass seedlings were susceptible to grasshoppers, crickets,
corn flea beetle, Chaetocnema pulicaria Melsheimer and
other insects, particularly when these insects inhabited
pre-existing vegetation killed for switchgrass establish-
ment. They reported that while not labeled at the time,
an in-furrow soil insecticide (carbofuran) provided con-
sistent advantages in establishment (Fike et al., 2006). In
addition, a report from Nebraska suggested that switch-
grass seedlings were susceptible to chinch bug, Blissus
leucopterus leucopterus (Say) and showed characteristic
reddening of the leaves in the greenhouse; however, nei-
ther nymphal infestations nor damage was observed in the
field (Ahmad et al., 1984). These data provide hints on the
types of pests that will challenge switchgrass. However,
these data have been collected over a limited geographic
area and yield losses due to pests have not been quantified.
Collecting these data will enable potential pest problems
to be compared between different biofuel crops across
varying geographic contexts.
Switchgrass genotypes show considerable variability in
insect susceptibility, suggesting the opportunity to breed


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C Institute of Zoology, Chinese Academy of Sciences, Insect Science, 17, 220–236
224 D. A. Landis & B. P. Werling
for resistance or discover resistance genes that may be use-
ful in molecular-based breeding programs. In Hawaii, the
‘Alamo’ cultivar of switchgrass proved among the most
resistant of all grass species tested to the yellow sugar-
cane aphid, Sipha flava (Forbes) (Miyasaka et al., 2007).
Dowd and Johnson (2009) report that seedlings of ‘Trail-
blazer’ and older ‘Blackwell’ plants were among the most
resistant to feeding by the fall armyworm, Spodoptera
frugiperda (J.E. Smith). Switchgrass accessions collected
from the field proved widely divergent, with some readily
fed upon by S. frugiperda and others causing high mortal-
ity in 2 days. Thus, both cultivated and wild populations
may contain a genetic variation that can be mined for
desirable traits.
Biofuel crops may also provide habitat for natural en-
emies. For example, tussock-forming grasses such as
switchgrass are known to improve habitat for a variety
of beneficial insects (Thomas et al., 1991). Frank et al.
(2008) tested the attractiveness of switchgrass and other
native plants to foliar and ground-dwelling insect natu-
ral enemies, planting them in conservation strips on golf
courses (Frank & Shrewsbury, 2004). These conservation
strips increased predator, parasitoid and alternate prey
numbers versus controls, primarily within 4 m of the
strip. Predation of black cutworm larvae, Agrotis ipsilon
(Hufnagel) was also higher in treatments containing
conservation strips. Similarly, plantings of other native,
tussock-forming grasses including big bluestem, Andro-
pogon gerardii Vitman and Indiangrass, Sorgastrum nu-
tans Nash, enhanced natural enemy abundance and pre-
dation of pests in potato over short distances (Werling,
2009). In a 2-year study examining carabid communities
in switchgrass, corn and sweetgum, Liquidambar styraci-
flua L. plantations, Ward and Ward (2001) consistently
captured more carabids in corn and switchgrass. However,
switchgrass had the highest mean species richness (12.1
and 6.5 species/year) and greatest mean diversity (prod-
uct of richness and evenness) of carabids with 29 species
in total collected. Most of these were common agricul-
tural species; communities were dominated by Harpalus
pennsylvanicus Dej. in one year and Anisodactylus furvus
LeConte in another. Menalled et al. (2001) studied the
carabid community in switchgrass and mixed alfalfa,
Medicago sativa L. and timothy, Pheleum pratense L. fil-
ter strips adjacent to soybean. Overall, carabids were more
abundant and diverse in switchgrass. Moreover, weed seed
removal was significantly higher in the switchgrass fil-
ter strips and was positively correlated with an increased
abundance of seed-feeding carabids in this habitat. In a
concurrent study, Carmona et al. (1999) reported signifi-
cantly greater abundance of the seed-feeding field cricket,
Gryllus pennsylvanicus Burmiester in the switchgrass
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C Institute of Zoology, Chinese Academy of Sciences, Insect Science, 17, 220–236
versus alfalfa/timothy strips or soybean. Overall, multi-
ple studies suggest that switchgrass may support abundant
populations of some natural enemies that could potentially
reduce biomass losses to pests.
Most of the above studies were not constructed to
compare arthropod communities between biofuel crops;
hence, comparisons relevant to making choices among
candidate biofuel crops are often not made. To ad-
dress this, Gardiner et al. (2010) contrasted the benefi-
cial arthropod communities in three biofuel crops (corn,
switchgrass and mixed prairie) to test the hypothesis that
more diverse plant communities would support increased
levels of beneficial arthropods. They found that most gen-
eralist predators and bees were either more abundant or di-
verse in the perennial and floristically more diverse grass-
lands than in corn. Bee communities were more species-
rich in switchgrass (n = 55 spp.) and significantly more
abundant in switchgrass than corn in early- to mid-season
samples. This approach allowed the arthropod communi-
ties inhabiting different biofuel crops to be directly com-
pared, suggesting that perennial grass systems may favor
a variety of beneficial arthropods over annual crops like
corn.
Miscanthus
Miscanthus × giganteus (hereafter miscanthus) is a hy-
brid grass that has been used for bioenergy production
in Europe since the 1960s and was recently introduced
to the US (Lewandowski et al., 2003). It is a triploid
(3×) hybrid produced by crossing M. sinensis (Thunb.)
(2×) with M. sacchariflorus (4×). Miscanthus provides
an example of the role that insect-vectored plant viruses
might play in influencing biomass production and pro-
duction of other crops. In Europe, few pests have been re-
ported that directly damage miscanthus; however, Huggett
et al. (1999) reported that the corn leaf aphid, Rhopalosi-
phum maidis Fitch was able to colonize miscanthus in the
greenhouse and was most fecund on established plants.
In addition, R. maidis successfully transmitted Barley
Yellow Dwarf Virus (BYDV) to miscanthus and symp-
toms were expressed. In contrast, the bird cherry-oat
aphid, Rhopalosiphum padi L. was unable to complete
development on miscanthus and BYDV transmission was
not demonstrated. They conclude that widespread produc-
tion of miscanthus may result in a large reservoir of BYDV
in the landscape and pose a threat to other sensitive crops,
particularly as the perennial miscanthus could represent a
bridging host for R. maidis from the time they leave cereal
crops in mid-summer and colonize newly planted cereals
in the fall.

C 2010 The Authors

Preliminary investigations have also examined the po-
tential conservation value of miscanthus for arthropods.
A wide variety of invertebrates inhabit miscanthus stands.
In one of the few studies on the arthropod commu-
nity of miscanthus, Semere and Slater (2007) sampled
ground beetles, butterflies and arboreal invertebrates in re-
establishing miscanthus stands following rhizome harvest
in England. In these 2–3-year-old, open canopy stands,
the miscanthus was only 53–265 cm tall and weed cover
was high (41%–96%). In contrast, established miscant-
hus stands may reach 4 m in height (Lewandowski et al.,
2003) and have closed canopies. As such, they noted that
the insect community was as much influenced by the weed
cover as the crop. Ground beetles were about as abundant
in miscanthus as in uncropped field margins, while butter-
flies were three-fold more abundant in field margins. The
abundance of arboreal invertebrates varied by taxa, but
was generally greater in field margins than in miscanthus
(Semere & Slater, 2007). Bellamy et al. (2009) studied
the bird community of miscanthus and winter wheat in
England and sampled the invertebrate community as a
potential food source using pitfall and sweep sampling.
These were also relatively young miscanthus stands in
their first to fifth growing seasons. In winter pitfall sam-
ples, overall invertebrate abundance did not differ between
the crops. In the spring samples, there was a significantly
greater abundance of foliar insects in wheat. The abun-
dance of other taxa was generally similar between the
crops, with the notable exception that Collembola were
more abundant in miscanthus. These studies suggest that
newly establishing miscanthus (with its open canopy and
weedy understory) may support relatively abundant pop-
ulations of some (e.g., carabids and Collembola) but not
all, arthropod taxa. These studies also highlight the im-
portance of expanding experimental treatments so that the
conservation benefits of competing biofuel crops can be
compared.
Relatively little has been reported regarding the use of
miscanthus by beneficial insects. In Japan, M. sinensis
has been used to provide overwintering and summer aes-
tivating sites for lady beetles in an attempt to enhance
their activity in nearby alfalfa fields (Takahashi, 1997).
In the eastern US, ornamental M. sinensis is attacked by
the miscanthus mealybug, Miscanthiococcus miscanthi
(Takahashi). Gordon and Davidson (2008) reported M.
miscanthi as a new prey record for the coccinellid Hy-
peraspis paludicola Schwarz and range expansion of the
coccinellid feeding on mealybug as far north as Washing-
ton, DC. This scarcity of information on natural enemies
and miscanthus points to a need to collect more data. This
will enable entomologists to evaluate the potential impacts
of this crop on biocontrol in agricultural landscapes.

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Arthropods and biofuel crops 225
Reed canary grass
Reed canary grass, Phalaris arundinacea L. is a
Eurasian and North American native plant that is used
as a forage and biomass crop in parts of Europe
(Landstrom et al., 1996). In the US, only a few populations
from Ontario are known to predate European settlement
and are considered native and non-invasive (Lavergne &
Molofsky, 2004). In contrast, non-native invasive geno-
types of reed canary grass now occur throughout much
of the US and Canada, likely as the result of multiple in-
troductions of European genotypes as a forage crop plant
and more recently for phytoremediation and wastewater
treatment (Lavergne & Molofsky, 2004). Reed canary
grass is being considered as a potential biomass crop
in the US (Perlack et al., 2005); however, others have
questioned its use given its invasive status (Raghu et al.,
2006).
Reed canary grass hosts a variety of pests and beneficial
insects, suggesting that comparing different biofuel crops
will require an accounting of both costs (e.g., pest damage)
and benefits (e.g., natural pest suppression) produced by
the arthropods they support. In Europe, reed canary grass
and other forage crops are reported to be attacked by a
variety of insects including the larvae of chafer beetles,
Melolontha melolontha L., wireworms, Agriotes spp., and
leather jackets Tipula spp. (Tscharntke & Greiler, 1995).
Semere and Slater (2007) also reported that reed canary
grass was infested by green peach aphid, Myzus persi-
cae (Sulz.) in England, with infestation levels reaching
20%. No yield losses were noted and BYDV symptoms
were absent. They also sampled ground beetles, butter-
flies and arboreal invertebrates in reed canary grass fields
and compared abundance and diversity to that found in
miscanthus. Reed canary grass harbored marginally lower
numbers of carabids, and butterfly abundance was two-
fold less than in miscanthus. There was no difference in
the diversity of butterflies between the two crops. Arbo-
real taxa were generally less abundant in reed canary grass
with the exception of Hemiptera populations, which were
more abundant (Semere & Slater, 2007).
Insect herbivores can use multiple host plants, creating
the opportunity for pests from one habitat to spill over into
other crops. For example, in the US, cereal leaf beetle,
Oulema melanopus L. is a pest of small grains. Cereal
leaf beetle larvae are known to feed on reed canary grass
(Wilson & Shade, 1966) and both cereal leaf beetle and
the frit fly, Oscinella frit L. have been reported as pests of
reed canary grass where it is used in wastewater treatment
facilities (Byers & Zeiders, 1976), suggesting the potential
for increased production of reed canary grass to cause pest
spillover onto grain crops. Thus, biofuels could provide
226 D. A. Landis & B. P. Werling
habitats for mobile pests that can move into other crops
and cause damage.
Reed canary grass is an invasive species that can spread
from plantings to other habitats, out-competing native
plants and reducing the diversity of associated arthro-
pods. For example, wetlands that are invaded by reed
canary grass show decreased plant diversity (Schooler
et al., 2006). This in turn has been related to a decrease in
moth species richness (Schooler et al., 2009). If produc-
tion of reed canary grass as a biofuel crop causes increased
invasion into natural wetlands, there could be negative im-
plications for native biodiversity. This example suggests
that if not responsibly used, novel biofuel plants could
become invasive and degrade native plant communities
and the arthropods that depend on them.
Mixed prairie
Tilman et al. (2006) proposed that a high-diversity,
low-input biofuel cropping system based on native US
tallgrass prairies may have a series of benefits over so-
called low-diversity high-input systems such as corn.
Specifically, these systems could be grown on marginal
soils with minimal inputs of fertilizer. As a result, little
carbon would be expended in their production, making
these feedstocks carbon-negative. Consequently, on low-
fertility soils, high-diversity low-input systems may have
nearly the same energy output as low-diversity high-input
systems and may be more economical to produce (Zhou
et al., 2009).
A complete review of insects in native prairie systems
is beyond the scope of this manuscript. Rangeland en-
tomology has been previously reviewed by Watts et al.
(1982), and Whiles and Charlton (2006) provide an ex-
cellent review of the ecological significance of arthropods
in tallgrass prairies. Here we focus on insect communities
in reconstructed grasslands, specifically on management
factors relevant to their utility as biofuel crops.
Mixed prairie could produce conservation benefits
for multiple arthropod taxa. In nearly all cases, bio-
fuel crops based on prairie communities would represent
prairie reconstructions not restorations, as they would
be planted on lands previously cleared for agriculture
or forestry. A number of studies have compared the in-
sect fauna of remnant and reconstructed prairies. Arthro-
pod species richness, diversity, or both are frequently
higher in remnant versus reconstructed prairies. This has
been shown for butterflies (Debinski & Babbit, 1997;
Shepherd & Debinski, 2005), grasshoppers (Bomar,
2009), Collembolla (Brand & Dunn, 1998), and mixed
taxa (Panzer et al., 1995). Larsen & Work (2003) showed
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C Institute of Zoology, Chinese Academy of Sciences, Insect Science, 17, 220–236
that carabid abundance and species richness was signifi-
cantly higher in reconstructed versus remnant prairies.
Other studies have compared arthropod communities
between prairies and other habitats. For example,
Larsen et al. (2003) showed that carabid beetles
were more abundant, species-rich and diverse in
either reconstructed or remnant prairies when com-
pared to woodlands or agricultural croplands, and that
prairies contained a higher percentage of specialist
species. Hopwood (2008) showed that roadsides re-
stored with native grassland plants supported a sig-
nificantly greater abundance and species richness of
bees compared to weedy unrestored roadsides. Finally,
Gardiner et al. (2010) found that bees were significantly
more abundant in mixed prairie than corn in early-mid
season samples. Overall, it can be concluded that a vari-
ety of arthropod taxa readily use reconstructed prairies.
Research suggests that the conservation benefits of
prairie are contingent on other factors, including land-
scape context and site management. Stoner and Joren
(2004) showed that insect communities in remnant prairies
were strongly affected by land management practices and
to a lesser degree by landscape factors. In general, plant
species richness declined with increasing intensity of
management from hay production to moderate- and high-
intensity grazing. In turn, plant community composition
explained the greatest amount of variation in Orthoptera
and Lepidoptera communities. In contrast, curculinoid
communities were equally influenced by blooming flower
density (which was only slightly influenced by manage-
ment) and landscape factors, while predator communities
were primarily influenced by landscape variables. Other
studies highlight the impact of site-scale variables on
arthropod communities. For example, Larsen and Work
(2003) found that carabid diversity in prairies declined
with time since burning, and Gardiner et al. (2010) found
that coccinellid diversity was positively correlated with
floristic diversity in reconstructed mixed prairies. These
studies demonstrate that the community-level impacts of
prairie-based biofuel production are likely to be very com-
plex and influenced by both within-site management and
landscape structure. Thus, the conservation benefits of
biofuels will vary between differently managed fields of
the same crop and in different landscapes.
Arthropod communities can in turn interact to influence
the structure and productivity of grasslands via complex,
indirect interactions. Schmitz (2008) showed that preda-
tors could influence the long-term biomass productivity
of grassland mesocosms by causing changes in herbivore
behavior. Specifically, the presence of sit-and-wait preda-
tors changed grasshopper behavior, forcing them to feed
on non-preferred plants. This resulted in slightly increased

C 2010 The Authors

plant diversity but significantly less overall biomass, per-
haps due to decreased N mineralization rates driven by
changes in litter input. This study shows that long-term
productivity of grassland-based biofuel crops is likely to
strongly depend on how arthropod communities interact
with plant diversity and overall management practices.
Furthermore, indirect interactions among insect species
could produce impacts on biomass that are not predictable
from studies that focus on individual insect herbivores and
plants. Thus, it will be important to complement lab stud-
ies of biofuel pests with field studies where herbivory of
biofuels is examined in its community context.
Arthropods and woody biofuel crops
Short-rotation woody crops
Use of short-rotation willow, Salix spp. and poplar, Pop-
ulus spp. plantations for biomass production, and more
recently phytoremediation, has a long history in northern
Europe (Perttu, 1995, 1999; Rowe et al., 2009). In the
US, clones of poplar (Dickmann et al., 2001) and willow
(Abrahamson et al., 1998; Volk et al., 2006) selected for
fast growth are planted at high densities and periodically
harvested (coppiced) promoting regrowth. The resulting
biomass can be used for direct energy generation in a
co-firing facility or used in production of liquid biofuel.
The biology and management of insect pests in North
American short-rotation hardwood systems were recently
reviewed by Coyle et al. (2005). They provide detailed life
histories for 32 insects attacking poplar, willow, sweetgum
and sycamore plantations in North America. They also
suggest general guidelines for reducing insect damage
in intensive plantations which include: (i) use of resistant
cultivars; (ii) using polycultures of different cultivars; (iii)
creating landscape mosaics of smaller plantings rather
than large monocultures; and (iv) maintaining high natural
enemy-to-pest ratios.
Both poplar and willow are attacked by a wide va-
riety of species and guilds of herbivorous insects. The
cottonwood leaf beetle, Chrysomela scripta (Fabricius) is
the most widespread and significant of the more than
300 insects and mites know to feed on poplar in the
US (Mattson et al., 2001). It is particularly damag-
ing to young trees. Poplar is also attacked by several
other important defoliators, sap feeders, and stem borers
(Coyle et al., 2005). Planting low-susceptibility clones
is a primary tactic to avoid insect damage in poplar.
Mattson et al. (2001) provide information on the suscepti-
bility of over 90 clones to C. scripta, spotted poplar aphid,
Aphis maculatae Oestlund, forest tent caterpillar, Mala-

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Arthropods and biofuel crops 227
cosoma disstria Hübner and tarnished plant bug, Lygus
lineolaris (Palisot de Beauvois). As cottonwood produc-
tion expands, new arthropod pests are still emerging. For
example, the cottonwood leafcurl mite, Tetra lobulifera
(Keifer) was only recently reported as a significant pest
of cottonwood (Coyle, 2002).
Willow is attacked by a variety of herbivores, and re-
search with willow herbivores provides an example of how
experiments can be used to streamline efforts to screen for
resistance. The imported willow leaf beetle, Plagiodera
versicolora (Laicharting) is the most economically dam-
aging pest of willow in the eastern US (Wade & Breden,
1986) and is capable of causing complete defoliation. Wil-
low is also attacked by a variety of Saliaceae specialist
and generalist defoliators (Coyle et al., 2005). Nordman
et al. (2005) screened 19 willow and 6 poplar clones
for resistance to defoliating insects and used multivariate
statistical approaches to analyze patterns of resistance.
They found significant correlations in the feeding pat-
terns among insects that could speed screening programs.
For example, they report that nearly all the differences
among clones of the seven insects could have been in-
ferred from the feeding patterns of just three generalist
species: Popillia japonica Newman adults, Nymphalis an-
tiopa L. larvae, and adults of either specialist, Polydrusus
impresifrons (Gyllenhall) or Crepidodera nana Say.
Labrecque and Teodorescu (2005) compared the field
performance of 10 willow and 2 poplar clones against
insect and disease attack in Quebec, Canada. They ob-
served P. versicolora, Disonycha alternata Illiger, Cal-
ligrapha multipunctata bigsbyana Kirby, Empoasca fabae
Harris, Janus abbreviates (Say) and Tuberolachnus
salignus Gemelin feeding on selected willow clones. In
addition, P. versicolora, D. alternata and Chaitophorus
populicola Thomas were occasionally observed on poplar
clones. In US tests, willow varieties with S. viminalis in
their background have been severely damaged by potato
leafhopper, Empoasca fabae Harris (Volk et al., 2006).
Willows and poplars may also support beneficial arthro-
pod communities and provide food for birds. Reddersen
(2001) suggested that the early and copious blooming of
willow may be important for flower-visiting insects in
Denmark. Sage and Tucker (1998) recorded over 120 in-
vertebrate species in the canopy of willows and poplar
in Europe and 45 species of ground-dwelling carabid and
staphylinid beetles. Cunningham et al. (2004) reported
greater species richness and diversity of butterflies at the
boundary of willow plantations versus arable land con-
trols. Sage et al. (1994) recorded 14 species of butterflies
in the margins of short-rotation plantations, with most
representing relatively common and widespread species
(Rowe et al., 2009). In studies of bird use of short-rotation
228 D. A. Landis & B. P. Werling
willow, Sage et al. (2006) characterized the plantings as
often weedy and insect-rich habitats. They found that nest-
ing birds used the willows as foraging sites and insect re-
mains were found in the feces of nestlings (Sage & Tucker,
1997; Sage & Tucker, 1998).
Biomass harvest from forests
In the US, woody biomass can be harvested from
forests in a variety of forms. Primary sources in-
clude logging residues from conventional harvesting
or land clearing operations and removal of excess
biomass in thinning or fuel reduction operations. Sec-
ondary sources include mill residues and pulping liquors,
with tertiary sources being urban wood residues from
tree-trimming and harvest or construction materials
(Perlack et al., 2005). Here we focus on the implications
of the harvest of primary woody biomass for bioenergy on
arthropod communities. This literature provides examples
of how within-site management can alter habitat structure
to affect arthropod communities.
Dead wood, including both coarse and fine woody de-
bris, is an essential resource that supports insect bio-
diversity in forest systems (Hanula et al., 2006). Re-
moving this debris for biofuels could affect species
that depend on this resource. For example, in Europe
saproxylic insects that feed on dead and decaying wood
are a highly threatened group (Davies et al., 2008).
Invertebrates make up nearly half (433/985) of the
rare to endangered plant, animal and fungi species in
Sweden and many of these depend on the presence of
dead wood (Berg et al., 1994). However, due to changes
in forest management, dead wood has declined in many
forest ecosystems (Fridman & Walheim, 2000; Norden
et al., 2004). Jonsell et al. (2007) showed that as many
as 22 European red-listed species utilize fine woody de-
bris as their primary habitat. They found large differences
among wood species and some among debris size classes.
Overall, they conclude that the fine woody debris from
some species, for example spruce, could be harvested for
bioenergy with less impact on invertebrate biodiversity
than other species.
Other studies suggest that the way in which trees are har-
vested could affect biocontrol of tree pests. Specifically,
Hedgren (2007) showed that in Sweden stumps serve as
habitat for a diversity of mostly harmless bark beetles
as well as their predators and parasitoids. In contrast to
normal low-cut stumps, high-cut stumps intended for in-
sect conservation purposes were favored by parasitoids
with the density of three species significantly increased
in their presence. By conserving habitat for natural ene-
Journal compilation 
C Institute of Zoology, Chinese Academy of Sciences, Insect Science, 17, 220–236
mies, high-cut stumps may favor biocontrol of destructive
bark beetles.
The treatment of remaining wood residues following
harvest also impacts forest arthropod communities. Re-
moval of slash from clear-cut areas reduced carabid bee-
tle but not lycosid spider abundance in the short-term
(Nitterus & Gunnarsson, 2006); however, 5–7 years af-
ter harvest the abundance and diversity of carabid bee-
tles was higher in slash-removal versus slash-remaining
sites (Nitterus et al., 2007). Slash-removal sites had in-
creased abundance of generalist species and a decline
in forest species. Finally, the spatial arrangement of har-
vested and unharvested areas can affect forest insects.
In a study of forest in which wood fuel was removed
to reduce fire risk, the damselfly, Argia vivida (Hagen)
preferred cleared fuel treatment areas during the day but
roosted in trees at night (Kortello & Ham, 2010). They
suggest that maintaining unmodified stands next to fuel
management areas would provide the best mix of habitats
to conserve this species. In contrast, a study manipulat-
ing coarse woody debris in southeastern US loblolly pine,
Pinus taeda L. forests showed no significant differences
in abundance, species richness or diversity of saproxylic
beetles (Ulyshen & Hanula, 2009). However, carabid bee-
tles were more species-rich and diverse in plots with log
inputs.
Genetic improvement of biofuel crops
Genetic improvement of biofuel crops will impact arthro-
pods in a variety of direct and indirect ways. Efforts
are underway to genetically improve many biofuel crops
(Vermerris, 2008) using techniques ranging from conven-
tional and molecular crop breeding to transgenic mod-
ification. Indeed, much of the corn currently grown in
the US includes trangenes from Bacillus thuringiensis
(Bt) (e.g. Pilcher et al., 2002) conferring resistance to
lepidopteran and coleopteran pests (Koziel et al., 1993;
Moellenbeck et al., 2001). Bt toxins have also been in-
corporated into some Populus varieties to protect against
coleopteran pests (Mattson et al., 2001). While questions
remain about the potential for transgenic plants to cause
insecticide resistance, secondary pest outbreaks and non-
target effects, if developed and deployed wisely they also
have potential to reduce insecticide use while promot-
ing beneficial natural enemies and pollinators (Kos et al.,
2009).
Improvement of biofuel crops to enhance processing
characteristics and energy yield could also cause a va-
riety of direct and indirect effects. For example, the
lignin content of plant cell walls reduces the efficiency of

C 2010 The Authors

cellulose breakdown during processing (Hisano et al.,
2009); low-lignin biofuels are thus considered a desirable
target for transgenic technology. Lignin is a product of the
shikimic acid pathway and modification of lignin quality
or quantity could alter production of other products of the
pathway, which include alkaloids and tannins important
in plant defense against insects (Bennett & Wallsgrove,
1994). Finally, even seemingly unrelated improvements to
biofuel crops such as incorporation of herbicide resistance
may alter insects and their management. For example,
several current biofuel crops (corn, soybean and canola)
include transgenes that confer herbicide resistance. This
alteration changes the ecology and management of these
crops in subtle ways that can be of significant impor-
tance to arthropods. For example, soybean producers that
grow glyphosate-resistant crops incur fixed costs (labor,
fuel, machinery etc.) to spray the herbicide. As such, the
relatively small additional cost to add an insecticide to
these applications may be seen as inexpensive “insurance”
against future insect attack. This situation has resulted in
an increase in insecticide applications against the soy-
bean aphid, Aphis glycines in midwestern US soybeans
(Olson et al., 2008) with potentially negative impacts on
natural enemies (Ohnesorg et al., 2009). While many im-
pacts of crop genetic improvement may be similarly dif-
ficult to anticipate, by collaborating with biofuel crop
developers, entomologists can help direct development of
sustainable biofuel cropping systems.
Landscape considerations
Meeting mandated targets for renewable ethanol in the
US will require harvesting biofuel feedstocks from agri-
cultural, grazing and forest lands (Perlack et al., 2005).
This will likely include increased production of traditional
biofuel crops, addition of new crops into the landscape
and increased harvest of biomass feedstocks from exist-
ing forest and rangelands. Doing so will cause changes
in landscape structure and landscape diversity that are
likely to have profound impacts on arthropods that are
both harmful and beneficial from a human standpoint.
Increasing monocultures?
Expansion of biofuel production could reduce land-
scape and plant diversity, creating monocultures that dis-
rupt predator–prey interactions. Specifically, pressures to
produce biomass could result in the production of one
or a few crops across entire agricultural landscapes, re-
ducing landscape diversity and biocontrol. For example,
Landis et al. (2008) showed that increased corn produc-

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C Institute of Zoology, Chinese Academy of Sciences, Insect Science, 17, 220–236
Arthropods and biofuel crops 229
tion for biofuels from 2005–2006 to 2007 in the US led
to a decline in agricultural landscape diversity in their
study area, resulting in reductions in predator abundance
and a loss of pest control services to neighboring crops.
Alternatively, Gardiner et al. (2009) suggest that increas-
ing grasslands in the same landscapes (thereby increasing
landscape diversity) could counter this effect and restore
biocontrol services. Similarly, a broad array of studies in
multiple regions suggest that natural enemy abundance,
diversity and pest control is greater in landscapes that
contain a mix of crop and non-crop habitats compared to
landscapes containing only annual crops (Bianchi et al.,
2006).
At the scale of a single crop, a wide variety of studies
similarly suggest that planting polycultures can reduce
pest problems and increase predator populations com-
pared to monocultures, although there are important ex-
ceptions (Andow, 1991). Accordingly, studies in mixed
prairie have shown that predator-to-prey ratios increase
with plant diversity (Haddad et al., 2009). Similar pro-
cesses may occur in forested landscapes. In a study com-
paring willow plantations to natural willow stands, Dalin
et al. (2009) found that while average density of the blue
willow leaf beetle, Phratora vulgatissima L. did not vary
between the habitat types during the 7-year study period,
the plantations showed greater temporal variation, result-
ing in outbreaks that were not observed in natural stands.
They suggest that generalist predators may reduce the po-
tential for outbreaks in mixed stands, and that larvae may
have a more difficult time finding a new host plant to feed
on after defoliating an individual tree in a mixed stand.
Implications for insect-vectored plant diseases
Increased cultivation of some biofuel crops is likely
to be associated with changes in the patterns of insect-
vectored plant diseases. For example, switchgrass stands
are reported to be attacked by the corn flea beetle, C.
pulicaria (Parrish et al., 1999) a vector of the bacterial
disease Stewart’s wilt, Erwinia stewartii raising the ques-
tion: will increased production of switchgrass alter the
epidemiology of this disease? Moreover, several poten-
tial biofuel crops including switchgrass and miscanthus
have been shown to be hosts for the Barley Yellow Dwarf
Viruses (BYDVs) (Garrett et al., 2004; Huggett et al.,
1999), a group of plant viruses vectored by a number of
grass-feeding aphids (Irwin & Thresh, 1990). As peren-
nial grasses, switchgrass and miscanthus may serve as per-
sistent reservoirs of virus, intensifying BYDV pressure on
annual small grains (A. Schrotenboer & C. Malmstrom,
personal communication). Insect vectors and viruses
230 D. A. Landis & B. P. Werling
harbored by biofuel crops may have important conse-
quences for natural communities as well. In California,
BYDV is now believed to have facilitated the invasion
and domination of perennial grasslands by exotic an-
nual grasses (Malmstrom et al., 2005). Healthy perennial
grasses are rarely invaded by healthy annual grasses; how-
ever, the presence of BYDV and aphid vectors reverses
the competitive relationship, allowing invasion and dom-
inance by annuals (Borer et al., 2007).
New landscape elements could influence
beneficial insects
The increase of any biofuel crop in the landscape will
almost undoubtedly change established patterns of arthro-
pod overwintering, movement, and their interaction with
host plants and each other. While North American studies
do not yet exist, it is easy to imagine that the addition
of perennial grasses as biofuel crops will provide over-
wintering sites for many arthropods due to their perennial
nature and well-developed litter layer. These characteris-
tics will likely alter arthropod decomposer communities
in the soil as well as influence their natural enemies at
higher trophic levels. Repeated harvest of perennial crops
without tillage may also alter soil structure, creating other
impacts on soil arthropods. Inclusion of woody biomass
crops in formerly annual crop landscapes will also change
predator communities in agricultural landscapes. For ex-
ample, Maredia et al. (1992b) showed that five species
of coccinellid beetles immediately adopted poplar planta-
tions when they were added to a research station as a short-
rotation biomass crop in a long-term experiment. Within
1 year, poplar treatments harbored the highest abundance
of the exotic seven-spotted lady beetle, Cocinella septem-
punctata L. (Maredia et al., 1992a) a common predator
in annual crop habitats. Colunga-Garcia and Gage (1998)
also showed that poplar habitats were readily utilized by
the multicolored lady beetle, Harmonia axyridis (Pallas)
which they implicated in the decline of several species of
native coccinellids. It seems clear from this single exam-
ple that the inclusion of new biofuel crop types in agricul-
tural landscapes will inevitably alter beneficial arthropod
communities in unexpected ways.
Other impacts of biofuels
Because arthropods are consumed by higher trophic
levels we can also expect significant shifts in the dynam-
ics of their consumers as well. Birds have already been
studied in biofuel crops to some degree; they respond to
both changes in habitat physical structure as well as to
Journal compilation 
C Institute of Zoology, Chinese Academy of Sciences, Insect Science, 17, 220–236
changes in food sources provided by arthropods (Sage
et al., 2006). A recent meta-analysis comparing verte-
brate (bird and mammal) abundance and diversity in bio-
fuel crops versus reference habitats shows consistent de-
clines when seminatural habitats are converted to biofuel
crops (Fletcher et al., 2010). Alternatively, the conver-
sion of current or former croplands to biofuel grasslands
(switchgrass or mixed prairie) has the potential to greatly
increase wildlife habitat in managed landscapes (Fargione
et al., 2009; Fletcher et al., 2010). Finally, the interaction
of arthropods and humans will undoubtedly be altered by
biofuel production. One could imagine landscapes where
biofuel crops increase landscape diversity to favor pollina-
tors and butterflies. Alternatively, if biofuel crops reduce
landscape diversity they may reduce these vital and aes-
thetically desirable amenities. Some of these impacts may
be so large that documenting them could help shape US
policy toward biofuels. For example, Landis et al. (2008)
have suggested that the design of future biorefineries is
likely to be a key decision point structuring future land-
scapes. If biorefineries are optimized for a single feed-
stock it is almost inevitable for that crop to increase dra-
matically within the supply zone of such a facility. They
suggest that biorefineries optimized to process multiple
feedstocks have the opportunity to help foster more di-
verse landscapes and resulting ecosystem services and
amenities.
Summary and conclusions
Our review suggests that expansion of biofuel cropping
systems in North America is likely to have extensive and in
many cases complex impacts on arthropod communities
in North American landscapes. Some of these impacts
will be beneficial, others harmful, and many will take
years to be fully realized. With expanded biofuel crop
production, existing arthropod pests of biofuel crops will
become more important and new pests will emerge. This
will require applied entomologists to develop novel inte-
grated pest management (IPM) systems for biofuel crop
pests. Management of biofuel crops, including crop se-
lection, agronomic practices, and harvest regimes will all
influence arthropod communities. In turn, arthropods will
impact the productivity and species composition of bio-
fuel cropping systems. Some of these processes have the
potential to produce landscape-level changes in arthropod
community dynamics and insect-vectored plant diseases.
In some cases, biofuel crops may harbor plant diseases
or insect vectors, potentially increasing damage to other
crops and even natural plant communities. Because of the
potential for long-range dispersal of insect vectors, such

C 2010 The Authors

impacts could have far-reaching effects and should be
used to inform selection of regionally appropriate biofuel
crops.
Beneficial arthropods will also be influenced by bio-
fuel crop habitats, altering the distribution of ecosystem
services they provide to the surrounding landscape. In-
sect natural enemies may be favored or disfavored by
biofuel crop choice. In landscapes currently dominated
by annual crops, the inclusion of new perennial biofuel
crops has the potential to increase natural enemy abun-
dance and diversity. Similarly, pollinators may be favored
by the addition of biofuel crops that provide food and
overwintering resources. In both cases careful selection
and addition of biofuel crops into agricultural landscapes
could increase the services these taxa provide. Arthro-
pods of conservation concern may be particularly vul-
nerable to habitat alteration and thus should be carefully
considered in selection of biofuel crops and in harvest
methods.
Finally, changes in arthropod abundance and their spa-
tial or temporal distribution in the landscape will have
impacts on consumers of insects at higher trophic levels,
potentially influencing their population and community
dynamics and producing feedbacks to arthropod commu-
nities. One might imagine a situation where introduction
of a biofuel crop increases insect abundance, which in turn
alters bird foraging and nesting success. This may in turn
increase avian predation of insects in the biofuel crop or
nearby habitats with effects that ramify through a variety
of food webs. Given that biofuel crops are still relatively
uncommon in North America, as they increase, we can
anticipate ‘predictably unpredictable’ shifts in arthropod
communities and the ecosystem services and functions
they support. We suggest that research on arthropod dy-
namics within biofuel crops, their spillover into adjacent
habitats, and implications for the sustainability of working
landscapes are critical topics for both basic and applied
investigations.
The research opportunities posed by novel biofuel pro-
duction systems are nearly endless and many could be
exceedingly important, perhaps to the point that the re-
sults of this research could alter the deployment of bio-
fuel crop technologies across the landscape. Given that
biofuel production systems are still being developed in
North America, entomologists have a unique opportunity
to consider how biofuel production may impact arthro-
pods, and may be able to initiate studies to document
these effects. Research is particularly needed to address
the potential impact of biofuel cropping on insects of con-
servation concern, the provision of key ecosystem ser-
vices such as pollination and pest suppression, and the
landscape level impacts of biofuel cropping on arthropod

C 2010 The Authors
Journal compilation 
C Institute of Zoology, Chinese Academy of Sciences, Insect Science, 17, 220–236
Arthropods and biofuel crops 231
communities and the organisms and ecosystem functions
they support.
Acknowledgments
Thanks to Lauren Bailey who assisted with literature
research and Bruce Robertson for helpful discussions
about the influence of arthropods on avian populations.
Mary Gardiner provided useful comments on earlier
drafts of the manuscript. This work was funded in part
by the DOE Great Lakes Bioenergy Research Center
(www.greatlakesbioenergy.org), which is supported by
the US Department of Energy, Office of Science, Office
of Biological and Environmental Research, through Co-
operative Agreement DE-FC02-07ER64494 between The
Board of Regents of the University of Wisconsin System
and the US Department of Energy. Additional support
was provided by the Michigan Agricultural Experiment
Station.
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Accepted November 2, 2009

C 2010 The Authors
 Insect Science (2010) 17, 237–244, DOI 10.1111/j.1744-7917.2009.01311.x

ORIGINAL ARTICLE

Hydrogen emission by three wood-feeding subterranean

termite species (Isoptera: Rhinotermitidae): Production
and characteristics

Yueqing Cao1,2 , Jian-Zhong Sun1,3 , Jose M. Rodriguez4 and Karmen C. Lee1

1 2
Coastal Research and Extension Center, Mississippi State University, Poplarville, Mississippi, USA, College of Bioengineering,
3
Chongqing University, Chongqing, China, School of the Environment, Jiangsu University, Zhenjiang, Jiangsu Province, China, and
4
Mississippi State Chemical Laboratory, Petroleum Production Division, Mississippi State University, Starkville, Mississippi, USA

Abstract Hydrogen emission by wood-feeding termites, Coptotermes formosanus,

Reticulitermes flavipes and Reticulitermes virginicus, was investigated upon a cellu-
losic substrate as their food source. The emission rates among the three species tested
were significantly different and R. virginicus demonstrated the greatest H2 emission at
4.78 ± 0.15 μmol/h/g body weight. In a sealed test apparatus, H2 emission for each ter-
mite species showed a quick increase at the initial incubation hours (3–6 h), followed by
a slower growth, possibly due to the feedback inhibition by gas accumulation. Further
investigation revealed that continuous H2 emission could be maintained by reducing the
H2 partial pressure in the sealed container. The bioconversion of cellulose to molecular
H2 by the subterranean termites tested could reach as high as 3 858 ± 294 μmol/g cellu-
lose, suggesting that the termite gut system is unique and efficient in H2 conversion from
cellulosic substrate.
Key words biofuel, hydrogen, methane, subterranean termite, symbiotic micro-organism

Introduction Hydrogen, in some references, is considered to play an

Termites are abundant social insects and cover about
two-thirds of the Earth’s terrestrial surface (Sanderson,
1996; Sugimoto et al., 1998). With the association of
gut symbionts, termites can decompose significant por-
tions of cellulose (74%–99%) and hemicellulose (65%–
78%) components of wood lignocellulose into glucose,
other hexose and pentose sugars, which are mainly fer-
mented to CO2 , H2 and acetate. Carbon dioxide and
hydrogen may then be released to the atmosphere or
possibly converted to CH4 and acetate through the activ-
ities of methanogenic and acetogenic bacteria in the ter-
mite gut system (Sugimoto et al., 1998; Ohkuma, 2003).
Correspondence: Jian-Zhong Sun, School of the Environ-
ment, Jiangsu University, Zhenjiang, 212013, China. Tel: +86
511 88796122; fax: +86 511 88790955; email: jzsun1002@
hotmail.com
intermediate role in linking the fermentative breakdown
of carbohydrate with methanogenesis and reductive ace-
togenesis (Ohkuma, 2003). Most of the phylogenetically
lower termites (characterized by having symbiotic protists
in their hindguts) show H2 –CO2 homoacetogenesis, indi-
cating that reductive acetogenesis dominates the hydrogen
sink reaction (Breznak & Switzer, 1986).
Hydrogen emission from termites is postulated to
be insignificant due to the fact that molecular H2 is
largely transferred from H2 -producing micro-organisms
to acetogens or methanogens within the termite’s hindgut.
This process internally consumes most of the produced
H2 (Breznak & Switzer, 1986; Brauman et al., 1992;
Brune, 1998; Sugimoto et al., 1998). However, hydrogen
emission by some termites has been reported and their
emission rates could vary from 122.0–5883.3 μmol/h/g
body weight (Sugimoto et al., 1998; Kawaguchi et al.,
2006; Inoue et al., 2007). Hydrogen and methane are


C 2010 The Authors 237
Journal compilation 
C Institute of Zoology, Chinese Academy of Sciences
238 Y. Cao et al.
simultaneously produced by termite gut symbionts which
include the protist inhabitants in the hindgut of the phy-
logenetically lower termites or the bacteria in the hindgut
of higher termites (typically without symbiotic protists in
their hindguts) (Taguchi et al., 1992; Breznak & Brune,
1994; Sugimoto et al., 1998; Leadbetter et al., 1999; In-
oue et al., 2007). It was also reported that H2 emission
rates and the ratio of CH4 /H2 varied with different termite
species (Sugimoto et al., 1998; Pester & Brune, 2007).
Wood-feeding termites, especially most of the lower ter-
mites, demonstrated a relatively higher H2 emission ca-
pability with a lower ratio of CH4 /H2 than higher termites
(e.g., soil-feeding and fungus-feeding termites) (Brauman
et al., 1992; Sugimoto et al., 1998; Pester & Brune, 2007).
For lower termites, hydrogen production is mainly at-
tributed to the dense population of cellulolytic protists
in the hindguts. Recently, two iron hydrogenase genes
were successfully identified from the protozoan, Pseu-
dotrichonympha grassii, in the hindgut of Coptotermes
formosanus, which directly clarified the molecular basis
for H2 production of this species (Inoue et al., 2007).
The efficacy in H2 emission largely depends on termite
species, food source, the symbiotic micro-organisms in
termite hindguts, as well as the mechanisms associated
with H2 production (Messer & Lee, 1989; Ebert & Brune,
1997; Schmitt-Wagner & Brune, 1999; Kawaguchi et al.,
2006; Tanaka et al., 2006). Effects of environmental, nu-
tritional and physiological stresses on termites would po-
tentially influence hydrogen/methane emission profiles
via symbionts in termite hindguts. Factors influencing the
intestinal micro-organisms or living conditions of termites
would also affect the gas emission from termites. Such
factors include chemical treatment to selectively remove
H2 - or CH4 -consuming symbionts (Messer & Lee, 1989),
termite diet (Rouland et al., 1993; Kawaguchi et al.,
2006), the atmosphere constituents in the headspace of
test containers (Tsunoda et al., 1993; Schmitt-Wagner &
Brune, 1999; Pester & Brune, 2007; Pester et al., 2007),
as well as the size of a test population (Tsunoda et al.,
1993).
Hydrogen emitted from termite guts could represent
a novel source of biohydrogen and a unique mechanism
in generating hydrogen from cellulose. Biohydrogen pro-
duction from lignocellulosic substrates is scarcely found
in other natural ecosystems (Sugimoto et al., 1998; Inoue
et al., 2007). The termite digestive system, which gen-
erates H2 in a significant amount from the degradation
of lignocellulose, has not received much attention for its
potential values in biohydrogen technologies. In the liter-
ature, most early investigations mainly focused on CH4
production from termite guts and its potential impact on
global warming (Fraser et al., 1986; Messer & Lee, 1989;
Journal compilation 
C Institute of Zoology, Chinese Academy of Sciences, Insect Science, 17, 237–244
Brauman et al., 1992; Darlington et al., 1997; Sugimoto
et al., 1998). Only a few termite species have been in-
vestigated for H2 emission (Ebert & Brune, 1997; Inoue
et al., 2007; Pester & Brune, 2007).
In this report, the profiles of H2 and CH4 emission were
established for the three wood-feeding lower termites,
C. formosanus, Reticulitermes flavipes, and Reticuliter-
mes virginicus. A modified CH4 /H2 index was introduced
to compare the characteristics of energy gas emission pat-
terns among the three termite species tested. Finally, the
effect of accumulated gas on continuous H2 emission
by a group of termites in a sealed container was also
investigated.
Material and methods
Termites
Worker and soldier termites of C. formosanus, R.
flavipes and R. virginicus were collected from Poplarville,
Mississippi, US. Each termite species was reared at 27–
28◦ C and > 90% RH in an individual plastic container
(31.8 × 25.6 × 9.7 cm, Tri-State Plastics, Latonia, KY,
USA) in the dark with moist sand and the originally
infested wood blocks.
Test apparatus
A wide-mouth glass bottle (diameter 4.3 cm, height
9.4 cm, inner volume 106 mL; VWR International, West
Chester, PA, USA, hereafter referred to as “apparatus”)
with a butyl rubber stopper (diameter 3.2 cm) was em-
ployed as test apparatus to hold termites. One piece of
Whatman No 4 filter paper (42.5 mm wide, Whatman,
Banbury, UK, > 99% cellulose) moistened with 150 μL
distilled water was placed in each apparatus to serve as
food and moisture for termites. Filter paper used in this
experiment was dried at 70◦ C for 1 h and weighed in-
dividually before and after the trial to estimate cellulose
consumption by the termites during the incubation period.
To test the air tightness of the apparatuses after being
sealed with a butyl rubber stopper, three bottles were ran-
domly sampled. One mL gaseous H2 was injected into
each of the three sealed empty bottles with an air-tight
pressure-lock syringe (VICI, Baton Rouge, LA, USA).
Then, 1-mL gas sample was immediately taken from each
bottle for H2 analysis. After incubation for an additional
72 h at 27◦ C, 1 mL gas was sampled again from each
of the three bottles. All samples were subjected to H2
analysis with gas chromatography (GC). Results showed

C 2010 The Authors

that no significant difference in H2 concentration was
detected (P < 0.001) at the beginning and the end of the
72 h incubation with 1 mL H2 . Thus, the experimental ap-
paratuses used were completely airtight with no detectable
H2 leakage.
Test procedure and gas sampling
Twenty worker termites (3rd instar or above) of each
species were weighed before use and placed in the appa-
ratus with a piece of wet filter paper, and then sealed with
a rubber stopper. The apparatus with termites was main-
tained in an incubator at 27◦ C. At each of the following
incubation intervals, 1, 3, 6, 24, 48, and 72 h, three appa-
ratuses were randomly selected for gas sampling. One-mL
gas samples from the headspace of each apparatus were
taken with a pressure-lock syringe. After gas sampling,
these three apparatuses were excluded from the test. Gas
samples were also taken from three sealed apparatuses
without termites to serve as base levels of H2 and CH4 .
All gas samples were subjected to GC analysis for H2 and
CH4 at Mississippi State Chemical Laboratory, Starkville,
MS, US.
Hydrogen emission with partial gas removal
Hydrogen emission by termites is reported to be con-
stant (Inoue et al., 2007). Our preliminary trial showed
that H2 emission did not continue after the H2 reached
a certain concentration in a sealed test container. There-
fore, we hypothesized that the accumulated gases had a
feedback inhibition on H2 emission and when the H2 equi-
librium was broken by removing some gas from the sealed
apparatus, it may possibly promote a continuous H2 emis-
sion by termites. To test this hypothesis, half volume of
gas, 53 mL, was removed with an air-tight pressure-lock
syringe from the headspace of each apparatus after 3 h
incubation, of which 1 mL was used for H2 analysis. Af-
ter incubation for an additional 3 h, another 1 mL gas
sample was taken from the same apparatus for H2 anal-
ysis. For comparison, the control treatment was set up
exactly the same except that no gas was removed at 3 h
incubation post-treatment. One-mL gas sample was taken
only at the end of the 6 h incubation period. At each in-
cubation interval, three replicates were sampled for each
termite species. Gas samples were analyzed with GC for
H2 . Total hydrogen emission of the two 3-h incubation
intervals was compared with that of the 6-h uninterrupted
incubation of the control group.

C 2010 The Authors
Journal compilation 
C Institute of Zoology, Chinese Academy of Sciences, Insect Science, 17, 237–244
Termites and biohydrogen 239
Hydrogen emission with modified test apparatus
To provide extra space for gas emission when emitted
gas by termites accumulated to a given level, the apparatus
was modified by connecting a vacuumed plastic gas sam-
pling bag (50 mL, Jensen Inert Company, Coral Springs,
FL, USA) to the apparatus with a syringe needle (51 mm,
22 gauge, Hamilton, Reno, NV, USA) through the butyl
rubber stopper. Four hundred C. formosanus workers with
two pieces of wet filter paper (Whatman No 4, 42.5 mm
wide), which served as termite food and moisture, were
pre-administered into each bottle prior to sealing. The
modified apparatus was then maintained in an incubator
at 27◦ C. After 48 h of incubation, 1 mL gas samples were
separately taken from both the bottle and the connected
gas bag with pressure-lock syringes. Total H2 production
of the modified apparatus was calculated by summing
the H2 from the bottle and the gas bag and compared with
that of the regular apparatus, in which 400 termite workers
were also used. Both the regular and modified apparatus
treatments were performed in triplicate.
Hydrogen conversion rate
The hydrogen conversion rate was defined as the capa-
bility for H2 emission (μmol) from each gram of filter
paper (cellulose) consumed by a termite species (μmol/g
filter paper). It was calculated by dividing the H2 emission
rate (μmol/h/g body weight) by the food consumption rate
(g filter paper/h/g body weight).
Gas analysis
Concentrations of H2 and CH4 in each gas sample were
analyzed using a Varian 3400 GC with N2 as the carrier gas
(25 mL/min, injector at 150◦ C, detector at 150◦ C, column
at 50◦ C). The GC was fitted with thermal conductivity
detector (TCD, Varian, Palo Alto, CA, US) and a steel
column (3 m × 3.2 mm) filled with a molecular sieve
13 × 45/60 mesh.
Data analysis
The effects of termite species on H2 and CH4 emis-
sion in the test apparatus at various sampling intervals
and the effect of partial gas removal from test appara-
tus on H2 emission for the three termite species tested
were analyzed using the analysis of variance (ANOVA)
(PROC MIXED model procedure, SAS, 2004; SAS Insti-
tute Inc., Cary, NC, US), respectively. Tukey’s Honestly
240 Y. Cao et al.
Significant Difference (HSD) test was used to separate
means at α = 0.05. The effect of the modified appara-
tus on H2 emission of C. formosanus, the air tightness of
the apparatus, and the food conversion rate were analyzed
with one-way ANOVA model.
Results
Emission of H2 and CH4
Hydrogen emission was detected for all three ter-
mite species tested and recorded at each observation
interval except the first incubation hour (Fig. 1). There
was a quick increase phase in H2 emission at the initial
3–6 h of incubation followed by a slow growth phase
after 24 h, which indicated a significant time depen-
dence on H2 emission (F 5,35 = 151.95, P < 0.001).
The recorded hydrogen concentration reached a plateau
at 10.07 ± 0.62 nmol/mL for C. formosanus, while for
R. flavipes and R. virginicus, the H2 concentration still
maintained a slow increase after 24 h of incubation. The
difference in the capability of H2 emission among the
three termite species tested was significant (F 2,35 = 29.66,
P < 0.001), where R. flavipes demonstrated the high-
est H2 concentration (15.63 ± 1.70 nmol/mL) at 72 h
(Fig. 1).
Methane (CH4 ) emission by R. flavipes and R. virgini-
cus was observed during the 72 h incubation period, where
18
H2 concentration (nmol/mL)
16
C. formosanus
14 R. flavipes
12
R. virginicus
10
8 CH4 /H2 index and the patterns of energy gas emission
6
4
2
0 three termite species tested. Each species demonstrated
0 10 20 30 40 50
Time (hour)
Fig. 1 H2 emission (nmol/mL) recorded in the test apparatus
during the period of 72 h incubation by Reticulitermes flavipes,
R. virginicus, and Coptotermes formosanus. Error bars indicate
1 s.e.m. The minimum level for quantitative determination of
H2 concentration is 0.1 nmol/mL, with the minimum detection
capability at 0.05 nmol/mL for H2 by the gas chromatography
machine used.
Journal compilation
CH4 concentration (nmol/mL)
60 C. formosanus
R. flavipes
R. virginicus
40
20
0
0 10 20 30 40 50 60 70
Time (hour)
Fig. 2 CH4 emission (nmol/mL) recorded in the test apparatus
during the period of 72 h incubation by Reticulitermes flavipes,
R. virginicus, and Coptotermes formosanus. Error bars indicate
1 s.e.m. The minimum level for quantitative determination of
CH4 concentration is 0.2 nmol/mL, with the minimum detection
capability at 0.1 nmol/mL for CH4 by the gas chromatography
machine used.
CH4 was released from termite guts simultaneously with
molecular H2 . However, no detectable CH4 emission was
recorded for C. formosanus during the 72 h observation
period (Fig. 2). Thus, the capability of methane emission
among the three termite species differed significantly for
the period we investigated (F 2,35 = 163.88, P < 0.001). In
contrast to the H2 emission profiles of the termite species
tested, the patterns of methane emission demonstrated a
steady growth in CH4 concentration during the 72 h incu-
bation period (Fig. 2). Time dependence was significant
on termite CH4 emission during the incubation period
(F 5,35 = 62.00, P < 0.001).
To define the characteristics of termite gas emission for
CH4 and H2 , a modified CH4 /H2 index (see the caption of
Fig. 3) was introduced. This index was distinct among the
a unique pattern in gas emission. As shown in Fig. 3,
60 70
the CH4 /H2 indexes for R. flavipes were characterized by
maintaining a growth trend at values much greater than
1 after 24 h incubation, suggesting that more CH4 was
emitted and accumulated in the test apparatus than H2 .
However, for R. virginicus, the CH4 /H2 index was much
lower than that of R. flavipes and fluctuated only from
0.4 to 1.2. This implied that R. virginicus had a similar
emission rate of CH4 and H2 at the same time. Since no de-
tectable CH4 was noticed for C. formosanus, the CH4 /H2
2010 The Authors 
C

C Institute of Zoology, Chinese Academy of Sciences, Insect Science, 17, 237–244
 Termites and biohydrogen 241

5 35

H2 emission ( mol/g body weight)

a Control
3 30
CH4 > H2 Partial gas removal
a
1 CH4 = H2
CH4 < H2 25
0
Index of CH 4/H2

-1 H2 only
20 b
b
-3
C. formosanus 15
-5
R. flavipes
R. virginicus a
10
-7 b

5
-9

0
-11
R. flavipes R. virginicus C. formosanus

0 10 20 30 40 50
Time (hour)
Fig. 3 Comparison of the profiles of H2 and CH4 emission
with the modified index of CH4 /H2 for Reticulitermes flavipes,
R. virginicus and Coptotermes formosanus during the period of
72 h incubation. A modified CH4 /H2 index from Sugimoto et al.
(1998) was introduced to define the characteristics of termite
gas emission for CH4 and H2 . Absolute concentration of CH4
and H2 in each gas sample was used for this calculation. It was
defined as follows: subtract H2 from CH4 when either H2 or CH4
concentration (nmol/mL) was zero; divide H2 by CH4 when the
60 70 80
Fig. 4 Effect of partial gas removal from test apparatus after
3 h incubation on continuous H2 emission by Reticulitermes
flavipes, R. virginicus and Coptotermes formosanus during a
total of 6 h incubation period (Letters represent mean separation
of H2 emission rates between gas removal and control, bars with
the same letter did not differ significantly at α = 0.05; PROC
MIXED model data analysis). Control represents no partial gas
removal from test apparatus. Error bars indicate 1 s.e.m.
a
H2 emission ( mol/g body weight)

3.0
concentrations (nmol/mL) of both H2 and CH4 were above zero;
name the CH4 /H2 index to be zero if there was no detectable H2
2.5
and CH4 . Therefore, more CH4 emission was measured than H2
b
when index was > 1 (denoted as CH4 > H2 ); CH4 emission was
2.0
equal to H2 when index was 1 (denoted as CH4 = H2 ); less CH4
was produced than H2 when index was from 0 to 1 (denoted
1.5
as CH4 < H2 ); only H2 was detected when index was negative
(denoted as H2 only).
1.0

index fell below zero, indicating that C. formosanus pre-
dominantly emitted H2 gas during the observation period
(Fig. 3).
Effect of gas accumulation on continuous H2 emission
When a portion of the gas volume was removed from
the test apparatus at 3 h (midway through the 6 h in-
cubation), the total H2 production during the 6 h in-
cubation period for each termite species was signifi-
cantly enhanced compared to the control groups with
no gas removal. The gas removal treatment from the
test containers resulted in a 39.3%, 40.9%, and 60.6%
more H2 emission by R. flavipes, C. formosanus, and
R. virginicus, respectively (Fig. 4). Clearly, gases accu-
mulated in a limited and sealed container significantly af-
fected continuous H2 emission by termites (F 3,12 = 32.12,
P < 0.001).
0.5
0.0
Regular Modified
Fig. 5 Comparison of the H2 emission rates of Coptotermes
formosanus between modified test apparatus (extra space avail-
able, i.e., a gas bag was connected with the test apparatus) and
regular test apparatus (without extra space). Letters represent
mean separation of H2 emission rates, bars with the same letter
did not differ significantly at α = 0.05; one-way ANOVA data
analysis. Error bars indicate 1 s.e.m.
Hydrogen emission of C. formosanus was 45.2% higher
in the modified test apparatus (extra space available for
gas emission to reduce the hypothesized feedback inhi-
bition by gas accumulation) than that in the regular test
apparatus (fixed and volume-limited without supplying
extra space) (F 1 = 11.32, P = 0.044) (Fig. 5).


C 2010 The Authors
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C Institute of Zoology, Chinese Academy of Sciences, Insect Science, 17, 237–244
242 Y. Cao et al.
Table 1 H2 emission and its conversion rate from cellulosic substrate by three wood-feeding termites, Reticulitermes flavipes, R.
virginicus and Coptotermes formosanus† .
Measurement
Food consumption rate (mg filter paper/h/g body weight)
H2 emission rate (μmol/h/g body weight)‡
H2 conversion rate (μmol/g filter paper)
†
Data were analyzed using PROC MIXED of SAS. Means in each raw followed by the same letter did not differ significantly at α =
0.05, Tukey’s Highly Significant Difference.
‡
The maximum emission rates during the 72 h incubation period were used.
Cellulose food consumption and H2 conversion rates
There was no observable change in termite feeding be-
havior during the 72 h incubation period and their survival
rates were maintained at 95%–100%. Food consumption
rates for the three termite species tested on the filter paper
substrate (> 99% cellulose content) ranged from 0.64–
1.26 mg/h/g body weight (Table 1).
Significant differences were found in the conversion
rates (F 2 = 18.73, P = 0.003) from the degradation of the
cellulosic filter paper to H2 among three termite species
tested, where R. virginicus showed the highest H2 con-
version rates at 3 858 ± 294 μmol/g cellulosic substrate
(Table 1). In a sealed apparatus environment, the highest
H2 emission rate for each of the termite species occurred
between the initial 3–6 h during the 72 h observation pe-
riod and it varied from 1.37–4.78 μmol/h/g body weight
depending on termite species (Table 1).
Discussion
Wood-feeding termites possess a great potential to re-
lease a significant amount of H2 as a byproduct from the
breakdown of cellulose in their guts. The hydrogen con-
version rates for the three termite species tested, in terms
of the amount of H2 released to the atmosphere, varied
from 2–4 mmol H2 /g cellulose (Table 1), which was far
lower than their intrinsic potential due to the internal H2
consumption by methanogenic and acetogenic bacteria
in their hindguts. Earlier reports showed that hydrogen
concentration remained high in the gut center and de-
creased sharply toward the periphery due to the H2 uptake
by methanogenic or acetogenic bacteria (Ebert & Brune,
1997; Brune & Friedrich, 2000), and as a consequence,
only a limited amount of H2 was emitted to the atmo-
sphere. Thus, the potential of hydrogen production and
hydrogen conversion rates by termites was greatly under-
estimated. In contrast to the termite gut system, most fer-
Journal compilation 
C Institute of Zoology, Chinese Academy of Sciences, Insect Science, 17, 237–244
C. formosanus R. flavipes R. virginicus
0.64 ± 0.06 b 1.04 ± 0.03 a 1.26 ± 0.14 a
1.37 ± 0.15 c 3.07 ± 0.23 b 4.78 ± 0.15 a
2 138 ± 88 b 2 941 ± 156 b 3 858 ± 294 a
mentative anaerobic bacteria or some H2 -producing bac-
teria isolated from termite guts showed 4–5 times higher
in the conversion rates from various mono-saccharides, or
starch substrates (Table 2) (Taguchi et al., 1993; Kumar
& Das, 2000; Oh et al., 2003; Lin & Lay, 2004; Kotay
& Das, 2007), where the hydrogen was collected via a
pure fermentative process without observable consump-
tion. However, the hydrogen production emitted by these
termites could be increased 5–7 times after an antibi-
otic treatment on their cellulosic diets (our unpublished
data). This study also confirmed that the termite gut sys-
tem could possibly maintain a continuous capability to
emit molecular H2 as long as the putative feedback inhi-
bition induced by the termite’s own gas emission could
be minimized. In the completely sealed test apparatus of
this study, maximum H2 emission rates for the three ter-
mite species tested were obtained within the first 3–6 h
of incubation (Table 1, Fig. 1). After that, the H2 con-
centration in the test containers increased slowly, possibly
due to the feedback inhibition effect from gas accumula-
tion, and eventually H2 concentration reached a plateau
(Fig. 1). Similar results were also reported in fermenta-
tive H2 production via anaerobic bacteria, in which the
yield of H2 was usually subjected to feedback inhibition
by H2 (Mousdale, 2008). Either by partially removing
gases from a constrained test apparatus or by connecting
a gas bag to serve as the extra space to the test apparatus
prior to the incubation has achieved a similar response –
enhancing the overall H2 production emitted by termites
(Figs. 4, 5). These investigations indicated that, by re-
ducing H2 partial pressure, the H2 emission capability of
termites could be maintained at a higher level. Thus, un-
derstanding why this H2 equilibrium occurs and how the
feedback inhibition takes place would potentially help us
break the H2 equilibrium for a continuous high yield of
H2 production released by termites.
Micro-organisms involved in hydrogen production in
the termite gut may have a potential to become robust can-
didates for biohydrogen processes. The micro-organisms

C 2010 The Authors
 Termites and biohydrogen 243

Table 2 Comparison of the substrate conversion rates by wood-feeding termites tested in this study and some fermentative bacteria
from references.

Maximum conversion rate

Organism Substrate Reference
(mmol H2 /g substrate)†

Termite
Coptotermes formosanus Cellulose 2.1 This report
Reticulitermes flavipes Cellulose 2.9 This report
R. virginicus Cellulose 3.9 This report
Bacteria isolated from C. formosanus
Clostridium beijerinckii AM21B Glucose 16.4 Taguchi et al., 1993
Starch 12.2 Taguchi et al., 1993
Fermentative bacteria
Enterobacter cloacae IIT-BT 08 Sucrose 17.5 (6) Kumar & Das, 2000
Clostridium pasteurianum Sucrose 14.0 (4.8) Lin & Lay, 2004
Citrobacter sp. Y19 Glucose 15.3 (2.76) Oh et al., 2003
Bacillus coagulans Glucose 12.7 (2.28) Kotay & Das, 2007
†
The conversion rates were determined by the maximum amount of H2 released to the atmosphere when termites fed on the cellulosic
filter papers during the 72 h incubation period. The figures in each parenthesis are cited from the original references, where the unit
used represents mol H2 /mol substrate.

that inhabit these lower termite digestive tracts, such as
protozoa and bacteria, contribute to cellulose and hemi-
cellulose digestion efficiency, and are most likely re-
sponsible for the subsequent emission of hydrogen and
methane (Breznak & Brune, 1994; Sugimoto et al., 1998;
Ohkuma, 2003). Some H2 -producing bacteria or hydroge-
nase genes have been isolated from termite guts (Taguchi
et al., 1992, 1993; Inoue et al., 2007). Although hydro-
gen is one of the byproducts during the breakdown of
lignocellulose in lower termites, to date, this character-
istic has received little attention. The termite gut as a
special bioreactor may hold the key to developing effi-
cient cellulose-based biohydrogen production because, in
most cases, nowhere else in nature would these unique
and efficient micro-organisms be found (Inoue et al.,
2007; Scharf & Tartar, 2008; Sun, 2008). Bioengineer-
ing techniques could also be helpful to construct an ef-
ficient H2 -producing strain for industrial-scale produc-
tion using these hydrogen-producing microbes or related
genes in the termite gut. Further research is also needed
to identify how termite guts function as the bioreac-
tors to facilitate cellulose degradation and subsequent H2
emission.
Acknowledgments
We thank E. J. Mallette for collecting and rearing termites
and D. St. Louis (Coastal Research and Extension Cen-
ter, Mississippi State University), C. Pounders (USDA-
ARS, small fruit unit, Poplarville, MS), and M. B. Layton
(Department of Entomology and Plant Pathology, Mis-
sissippi State University) for reviewing an early draft of
this manuscript. This work is supported by the Southeast
Regional SunGrant Program sponsored by US Depart-
ment of Transportation. Project Number: DTOS59-07-G-
00050 (subaward #: 101564). The paper was approved for
publication by the Mississippi Agriculture and Forestry
Experimental Station as Manuscript J-11425.
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Accepted November 11, 2009

C 2010 The Authors
 Insect Science (2010) 17, 245–252, DOI 10.1111/j.1744-7917.2010.01321.x

ORIGINAL ARTICLE

Hydrolysis of filter-paper cellulose to glucose by two

recombinant endogenous glycosyl hydrolases of
Coptotermes formosanus

Dunhua Zhang1 , Alan R. Lax1 , John M. Bland1 , Jiujiang Yu2 , Natalie Fedorova3,4
and William C. Nierman4
1
Formosan Subterranean Termite Research Unit, Southern Regional Research Center, ARS, USDA, New Orleans, 2 Food & Feed Safety
Research Unit, Southern Regional Research Center, ARS, USDA, New Orleans, 3 Department of Biochemistry and Molecular Biology, The
George Washington University School of Medicine, Washington, DC, and 4 The J. Craig Venter Institute, Rockville, MD, USA

Abstract Genes encoding for glycosyl hydrolases (GH) in multiple families were recov-
ered from an expression sequence tag library of Coptotermes formosanus, a xylophagous
lower termite species. Functional analyses of these genes not only shed light on the mecha-
nisms the insect employs to successfully use cellulosic materials as energy sources, which
may serve as strategic targets for designing molecular-based bio-pesticides, but also enrich
discoveries of new cellulolytic enzymes for conversion of biomass into biofuel. Our study
demonstrated that cellulose could be converted to glucose by two recombinant endogenous
glycosyl hydrolases (endo-β-1,4 glucanase in GH9 and β-glucosidase in GH1). While the
former cleaved cellulose to cellobiose and cellotriose, the resulting simple cellodextrins
were digested to glucose. Both of the Escherichia coli-expressed recombinant proteins
showed properties that could be incorporated in a glucose-based ethanol production pro-
gram.
Key words biofuel, cellulose digestion, endo-β-1,4 glucanase, β-glucosidase, termite

Introduction
The Formosan subterranean termite, Coptotermes for-
mosanus Shiraki, is one of the most destructive and
costly wood-feeding insects in many parts of the
world (Su & Scheffrahn, 2000; Lax & Osbrink, 2003;
Archicentre, 2003; Tsunoda, 2003). Clarifying the molec-
ular mechanisms that enable the wood-degrading insect
to utilize lignocellulose as a sole food source is thus a tan-
talizing area of research in which to explore the potential
of developing cellulolytic enzyme-specific biopesticides
Correspondence: Dunhua Zhang, Formosan Subterranean
Termite Research Unit, Southern Regional Research Cen-
ter, ARS, USDA, 1100 Robert E. Lee Boulevard, New Or-
leans, LA 70124, USA. Tel: (504) 286 4382; email: dunhua.
zhang@ars.usda.gov
(Zhu et al., 2005; Zhou et al., 2008) and optimizing re-
combinant cellulolytic enzyme production for biomasss
conversion (Ni et al., 2005, 2007a, 2007b; Todaka et al.,
2009).
Previously, we demonstrated that C. formosanus-
derived endogenous endo-β-1,4-glucanase, expressed in
Escherichia coli, can digest filter-paper cellulose releas-
ing predominantly cellotriose and cellobiose (Zhang et al.,
2009). This suggests that this salivary gland-abundant
cellulase (Nakashima et al., 2002) could be involved in
initial cleavage of cellulosic materials ingested by the
termite. The function of this cellulase also implies that
cellulose could be converted to glucose when an ad-
ditional β-glucosidase is present, without the aid of a
1,4-β-D-glucan cellobiohydrolase (cellobiohydrolase, ex-
oglucanase), which is thought necessary for effective bi-
ological hydrolysis of cellulose to glucose in fungal and
bacterial cellulolytic systems (Béguin & Aubert, 1994).


C 2010 The Authors 245
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C Institute of Zoology, Chinese Academy of Sciences
246 D. Zhang et al.
A β-glucosidase is known to be present in the salivary
gland and midgut of C. formosanus (Itakura et al., 1997;
Zhu et al., 2005). However, the gene sequence of the
putative β-glucosidase has not been analyzed and its cel-
lulolytic activity is unknown.
In this study, the endogenous β-glucosidase gene of
C. formosanus was cloned from a cDNA library of worker
termites and heterologously expressed in E. coli. The en-
zymatic activity of the recombinant β-glucosidase was
determined using cellodextrins and enzymatic end prod-
ucts of filter-paper cellulose catalyzed by a recombinant
endogenous endo-β-1,4-glucanase.
Materials and methods
Cloning of β-1,4-glucanase and β-glucosidase genes
The β-1,4-glucanase gene (accession No. EU853671)
was cloned as described previously (Zhang et al., 2009).
The primers used for cloning of β-glucosidase gene were
designed based on the sequence derived from a C. for-
mosanus expression sequence tag (EST) library (which
was made from a pool of normalized mRNA, including
different developmental casts; the EST data is under an-
notation). The full-length cDNA of β-glucosidase was
cloned from the termite worker rapid amplification of
cDNA ends (RACE)-ready cDNA using the gene-specific
primers and the SMART RACE cDNA Amplification kit
(Clontech, Mountainview, CA, USA). Resulting cDNA
clones were sequenced and analyzed using basic local
alignment search tool (BLAST) search and Vector NTI
program (Invitrogen, Carlsbad, CA, USA).
Production of recombinant proteins in E. coli
The coding sequences for the mature peptides of
endo-β-1,4-glucanase (Zhang et al., 2009) and β-
glucosidase (predicted by on-line program SignalP 3.0
Server: www.cbs.dtu.dk/services/SignalP) were poly-
merase chain reaction (PCR)-amplified using the fol-
lowing primer pairs: (i) 5 -CTAGCCATG GCT TAC
GAC TAC AAG ACA GTA CTG AAG A-3 (forward)
and 5 -CAGACTCGAG CAC GGC TGC CTT GAG
GAG ACC-3 (reverse) for endo-β-1,4-glucanase; (ii)
5 - CTAGCCATG GAT GAC GTC GAT AAC GAC
ACC CTT G -3 (forward) and 5 -CAGACTCGAG GTC
TCG GAA GCG CTC TGG AAT CTG-3 (reverse) for
β-glucosidase. Both forward primers flanked an Nco I
restriction site at the 5 end and reverse ones had an Xho
I site at the 5 end (underlined). Amplicons were gel-
purified and subjected to double digestion with Nco I and
Journal compilation 
C Institute of Zoology, Chinese Academy of Sciences, Insect Science, 17, 245–252
Xho I (New England BioLabs, Ipswich, MA, USA). Fol-
lowing further purification, both amplicons were cloned
into pET28a vector plasmids (Novagen, Madison, WI,
USA) at corresponding restriction sites. Recombinant
plasmids were propagated in E. coli NovaBlue cells (No-
vagen) and purified for sequence analysis.
Competent cells of E. coli Rosetta 2 (DE3) (Novagen)
were transformed with recombinant plasmids carrying
an endo-β-1,4-glucanase gene or β-glucosidase gene,
respectively, and selected on Luria-Bertani (LB) agar
supplemented with antibiotics (50 μg/mL kanamycin
and 34 μg/mL chloramphenicol). Resulting colonies
were propagated in LB broth containing the antibi-
otics for glycerol stocks and recombinant protein
production.
Actively growing colonies on LB agar (37◦ C, overnight)
were inoculated in LB broth with antibiotics and shake-
cultured at 250 r/p at 37◦ C for approximately 6 h.
Bacterial cells were pelleted by centrifugation and were
further propagated in 40 mL fresh LB broth with an-
tibiotics by shaking at 37◦ C until reaching optical den-
sity (OD600 ) of approximately 0.4–0.5. Protein over-
expression was induced by addition of IPTG ((isopropyl-
β-D-thiogalactopyranoside), at a final concentration of
0.5 mmol/L, to the bacterial cultures with continuous
shaking at 23◦ C for 16 h. The induced bacterial cells were
then aliquoted and pelleted by centrifugation. Total cell
proteins were extracted using 1× CelLytic B Cell Lysis
Reagent (Sigma, St Louis, MO, USA), which was diluted
with a buffer containing 20 mmol/L Tris, 0.5 mol/L NaCl,
pH 7.9. The cell lysates were clarified by centrifugation.
Target proteins were purified from the resulting super-
natants using His•Mag Agrose Beads (Novagen) in accor-
dance with the manufacturer’s instruction. Protein concen-
tration of each preparation was estimated with Bradford
Reagent (Sigma) using bovine serum albumin (BSA) as
the protein standard. Protein profiles of crude extracts and
purity of purified proteins were determined by sodium do-
decyl sulfate – polyacrylamide gel electrophoresis (SDS-
PAGE) using NuPAGE 4%–12% Bis-Tris pre-cast gel and
MOPS/SDS running buffer (Invitrogen).
Digestion of cellodextrins/filter paper with
the recombinant proteins
Cellodextrins used included cellobiose (99% purity,
Sigma), cellotriose (98% purity, Sigma), cellotetraose
(90% purity, Sigma) and cellopentaose (80% purity,
Sigma). Each was dissolved in 50 mmol/L sodium ac-
etate (pH 5.0) at concentrations of 10 mg/mL. An aliquot
of 100 μL of the cellodextrin was individually mixed with

C 2010 The Authors

5 μL (100 ng) of purified β-glucosidase. The mixture was
incubated at 37◦ C for 1 h. An aliquot of 1 μL of the reac-
tion was analyzed using thin layer chromatography (TLC)
described below.
Filter paper digestion was conducted by mixing 15 mg
of pre-cut filter paper (∼1–2 × 1–2 mm, Whatman #4)
with 200 μL 50 mmol/L sodium acetate buffer (pH 5.0)
containing 200 ng of purified endo-β-1,4-glucanase. The
mixture was incubated at 37◦ C for 24–48 h. An aliquot of
5 μL was analyzed using TLC. Additionally, an aliquot
of 50 μL of the reaction was mixed with 2.5 μL (50 ng) of
purified β-glucosidase and incubated at 37◦ C for 1 h. An
aliquot of 5 μL of the second reaction was also analyzed
using TLC.
Thin layer chromatography conditions were performed
as described previously (Zhang et al., 2009). Briefly, af-
ter spotting the samples on an TLC plate (Silica Gel 60,
EMD Chemicals Inc, Gibbstown, NJ, US), chromatogra-
phy was developed in a solvent of n-butanol/glacial acetic
acid/water (2 : 1 : 1) for 1.5 h. The plate, after drying
with a stream of warm air, was stained by spraying with
p-anisaldehyde reagent (ethanol : glacial acetic acid : sul-
furic acid : p-anisaldehyde = 9.0 : 0.1 : 0.75 : 0.75) and
heated in an oven at 110◦ C for 5–10 min. Reference sugar
standard, containing 10 μg each of glucose, cellobiose,
cellotriose, cellotetraose and cellopentaose, was included
in each TLC run.
β-glucosidase activity on cellobiose at different pH
and temperature
The optimal ranges of pH and temperature for
β-glucosidase activity on cellobiose were determined by
the following settings. Buffers with pH range from 3.8
to 7.8 were prepared using 100 mmol/L acetic acid per
100 mmol/L sodium acetate (pH 3.8–5.6) and 100 mmol/L
NaH2 PO4 per 100 mmol/L Na2 HPO4 (pH 6.2–7.8).
Aliquots of 100 μL cellobiose solution at concentration
of 5 μg/μL with different pH units, containing 200 ng of
β-glucosidase, were incubated at 37◦ C for 75 min. Fol-
lowing incubation, an aliquot of 2 μL for each reaction
was analyzed by the TLC method described above.
Five temperature assays were conducted between 29◦ C
and 60◦ C. Aliquots of 100 μL cellobiose solution at
5 μg/μL in 100 mmol/L sodium acetate buffer (pH 5.0),
containing 200 ng of β-glucosidase, were incubated at
different temperatures. At a 15-min interval an aliquot of
10 μL reaction was taken from individual reactions and
placed on ice until TLC analysis, the same method as
described above.

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C Institute of Zoology, Chinese Academy of Sciences, Insect Science, 17, 245–252
Coptotermes formosanus cellulases 247
Results
β-glucosidase gene structure
The complete cDNA of C. formosanus β-glucosidase,
as shown in Fig. 1, was the first of its kind cloned from
this insect (GenBank accession no.: GQ911585). The full-
length sequence consists of 1 745 bp excluding the poly-A
tail. The single open reading frame indicates that the trans-
lation starts at nucleotide 106 and ends at thenucleotide
1 593, encoding 495 amino acids. There is a predicted 17-
amino acid signal peptide. The 478-amino acid mature
peptide has a calculated molecular weight of 54.58 kDa
with isoelectric point (pI) of 4.77. Protein BLAST search
shows that the protein is similar to β-glucosidases in gly-
cosyl hydrolase (GH) family 1 and possesses correspond-
ing amino acid residues for catalysis and substrate binding
(Marana et al., 2001).
Expression of recombinant proteins
Production of recombinant endo-β-1,4-glucanase and
β-glucosidase in E. coli was achieved using IPTG induc-
tion. Apparently homogeneous recombinant proteins were
recovered from the crude extracts following affinity pu-
rification (Fig. 2). The endo-β-1,4-glucanase shows a dis-
tinctive band with molecular weight of ∼48 kDa while the
distinctive band for β-glucosidase has a molecular weight
of ∼ 56 Daltons on SDS-PAGE. From a 10 mL culture, ap-
proximately 150–200 μg purified proteins were obtained
for both endo-β-1,4-glucanase and β-glucosidase.
Enzymatic hydrolysis of cellodextrins and filter-paper
cellulose
The recombinant endo-β-glucosidase could hydrolyze
cellobiose, cellotriose, cellotetraose and cellopentaose
and generated only glucose as the end product as shown
in Fig. 3. Filter paper was not hydrolyzed by the en-
zyme even in a prolonged incubation (at 37◦ C for 4 days;
lane 5 of Fig. 4). On the other hand, the recombinant
endo-β-1,4-glucanase could hydrolyze filter-paper cellu-
lose and produced mainly cellobiose and cellotriose in a
24-h incubation at 37◦ C (Lane 2, Fig. 4). When the re-
action was kept at 37◦ C for another 24 h or longer, the
end products of the hydrolysis were mainly cellobiose
and glucose, and no cellotriose was detected (Lane 3,
Fig. 4). By addition of recombinant β-glucosidase to
the hydrolytic end products of filter paper catalyzed by
endo-β-1,4-glucanase (24 h, 37◦ C), only glucose was
248 D. Zhang et al.

Fig. 1 The complete cDNA and translated amino acid sequences of Coptotermes formosanus β-glucosidase. The 5 and 3 untranslated
sequences are in italic; the poly-A signal is underlined. The 17-amino acid signal peptide is highlighted in bold. Amino acid residues,
involved in catalysis and substrate binding, are underlined.


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C Institute of Zoology, Chinese Academy of Sciences, Insect Science, 17, 245–252
 Coptotermes formosanus cellulases 249

Fig. 3 Thin layer chromatography of cellodextrin hydrolytic

products by the recombinant β-glucosidase. In Lane 1, cel-
lodexrins, G1–G5, indicate glucose, cellobiose, cellotriose,
cellotetraose and cellopentaose, respectively; each loaded at ap-
proximately 10 μg. Lanes 2–5: hydrolytic products (at 37◦ C for
1 h) of G2, G3, G4 and G5, respectively (approximately 10 μg
of substrate equivalent were spotted).
Fig. 2 Sodium dodecyl sulfate – polyacrylamide gel elec-
trophoresis of recombinant endo-β-1,4-glucanase (A) and β-
R
glucosidase (B). A: Lane 1: molecular marker (SeeBlue Plus
2, Invitrogen, Carlsbad, CA, US); Lane 2: crude extract of re-
combinant endo-β-1,4-glucanase; Lane 3: purified endo-β-1,4-
glucanase. B: Lane 1: molecular marker (Invitrogen); Lane 2:
crude extract of recombinant β-glucosidase; Lane 3: purified β-
glucosidase. The gels were stained with SimpleBlue SafeStain
(Invitrogen).

detected in the reaction after 1-h incubation at 37◦ C

(Lane 4, Fig. 4).

β-glucosidase activity at different pH and temperature

With cellobiose as substrate, enzymatic conversion

to glucose by β-glucosidase was almost completed in
75 min at pH 5.0–6.6. Beyond this pH range the enzy-
matic activity decreased gradually (Fig. 5). The optimal
temperature range for the β-glucosidase activity is shown
in Fig. 6. For the complete conversion it took ∼75 min
at 29–37◦ C, 60 min at 42◦ C, 45 min at 48–54◦ C, and
60 min at 60◦ C. While the optimal temperature for the en-
zymatic activity ranged from 42◦ C to 54◦ C, the enzyme
was apparently unstable when the temperature reached to
60◦ C.
Fig. 4 Thin layer chromatography of hydrolytic products of
filter paper. Lane 1: cellodextrin standards, the same as depicted
in Figure 3. Lane 2: recombinant endo-β-1,4-glucanase with
filter paper (at 37◦ C for 24 h); Lane 3: the same reaction as Lane
2 but incubated at 37◦ C for 48 h; Lane 4: the end products of Lane
2 with addition of recombinant β-glucosidase and incubation at
37◦ C for 1 h; Lane 5: recombinant β-glucosidase with filter
paper (at 37◦ C for 4 days).


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250 D. Zhang et al.

Fig. 5 Thin layer chromatography of cellobiose hydrolytic

product by the recombinant β-glucosidase at different pH solu-
tions. Cellobiose was dissolved in 100 mmol/L sodium acetate
buffer (pH 3.8–5.6) or 100 mmol/L sodium phosphate buffer
(pH 6.2–7.8) at concentration of 5 μg/μL and incubated at 37◦ C
for 75 min. An aliquot of 2 μL was applied to the analysis.

Discussion

Lignocellulosic biomass stores energy in the form

of plant cell wall polymers (cellulose, hemicellulose
and lignin) and offers a renewable source of sug-
ars that can be converted to ethanol and other liq-
uid fuels (Rubin, 2008). Termites are considered as an
extremely successful group of wood-degrading or-
ganisms (Sugimoto et al., 2000). Extensive research
efforts have been made to determine the lignocellulose-
degrading/energy-conversion biocatalysts residing in ter-
mites and their symbiotic/mutualistic microbes, such
as Nasutitermes sp. hindgut microbiota (Warnecke
et al., 2007), protists of Reticulitermes speratus (Todaka
et al., 2007), an endosymbiont of C. formosanus protist
(Hongoh et al., 2008), and Reticulitermes flavipes host
and symbionts (Scharf & Tartar, 2008).
To identify and characterize the unique molecular-
processing mechanisms of C. formosanus, which enable
the termite to undergo caste differentiation and me-
tabolize wood lignocellulose, we have sequenced ap-
proximately 75 000 clones from a normalized C.
formosanus EST library (the annotation is ongoing).
Preliminary assessments show that genes encoding mul-
tiple families of glycosyl hydrolases were present in the
C. formosanus transcriptome as seen from other termite
species mentioned above, though sequence (codon) vari-
ations were observed. Most of the endogenous glycosyl
hydrolases are expressed predominantly in C. formosanus
salivary glands as revealed by quantitative reverse
Fig. 6 Thin layer chromatography of cellobiose hydrolytic
transcription-PCR (D. Zhang & A.R. Lax, manuscript in product by the recombinant β-glucosidase at time course from 0
preparation), suggesting that they may be directly involved to 75 min at different temperatures. Cellobiose was dissolved in
in wood lignocellulose digestion. 100 mmol/L sodium acetate buffer (pH 5.0) at concentration of
The demonstrative experiments conducted in 5 μg/μL and incubated at corresponding temperature settings.
this study provide evidence that the recombinant An aliquot of 2 μL was applied to the analysis.


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C Institute of Zoology, Chinese Academy of Sciences, Insect Science, 17, 245–252

endo-β-1,4-glucanase and β-glucosidase derived from
C. formosanus and produced in E. coli are active in
hydrolyzing cellulose to glucose. The endo-β-1,4-
glucanase produced mainly cellobiose and cellotriose
from the cellulose and could convert cellotriose into
cellobiose and glucose in a prolonged reaction. Although
endoglucanases are generally thought to hydrolyze
internal β-1,4-glycosidic bonds, the insect-origin
endo-β-1,4-glucanases seem to possess characteristic
properties of cellobiohydrolase, an exoglucanase, as
observed in others extracted/purified from a higher
termite (Nasutitermes walkeri; Schulz et al., 1986), a
lower termite (Reticulitermes speratus; Watanabe et al.,
1997) and a cockroach (Panesthia cribrata; Scrivener &
Slaytor, 1994). Recently, an endoglucanase (GH family
7), derived from an R. speratum symbiont and expressed
in Aspergillus oryzae, was shown to have similar prop-
erties (Todaka et al., 2009). The molecular mechanism
underlying this property is currently unknown. The
C. formosanus β-glucosidase, as first reported here in
recombinant form, hydrolyzed 2–5 units of cellodextrin
polymers but not cellulose. This property resembles
that of a recombinant β-glucosidase of Neotermes
koshunensis (Ni et al., 2007b), although differential
enzymatic activities were observed (D. Zhang & A.R.
Lax, manuscript in preparation).
The functions of most sequencing-derived cellulolytic
genes are predicted, and their enzymatic activities, sub-
strates and eraction end products have yet to be de-
termined. Actually, some cellulases may have altered
activities or even gained new functions (Davison &
Blaxter, 2005). A β-glucosidase (GH family 1) in
Cryptotermes secundus has apparently evolved from an
ancestral role of wood digestion to pheromonal commu-
nications (Korb et al., 2009) and a β-1,3-glucanases (GH
family 16) in Nasutitermes corniger (Bulmer et al., 2009)
and in Helicoverpa armigera (Pauchet et al., 2009) was
found to serve as an antimicrobial effector. Copper oxi-
dase (laccase) was reported to be responsible for the cuti-
cle sclerotization and tanining in Bombyx mori (see Yatsu
& Asano, 2009); however, in wood-feeding termites the
enzyme may be related to lignin oxidation or depoly-
merization (Scharf & Tartar, 2008). Therefore, functional
analysis of candidate lignocellulytic proteins in recom-
binant form in large quantities, as demonstrated in this
study, could reveal potential applications in the biofuel
conversion industry.
Further understanding of the efficient wood-
degrading/energy-conversion enzyme system of ter-
mites, especially the molecular mechanism of lignin/
crystalline microfibril depolymerization, could lead to
development of better enzyme cocktails with higher effi-

C 2010 The Authors
Journal compilation 
C Institute of Zoology, Chinese Academy of Sciences, Insect Science, 17, 245–252
Coptotermes formosanus cellulases 251
ciency in bacterial or yeast expression systems for green
energy production.
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Accepted December 3, 2009

C 2010 The Authors
 Insect Science (2010) 17, 253–264, DOI 10.1111/j.1744-7917.2010.01323.x

ORIGINAL ARTICLE

Identification of proteins involved in lignocellulose

degradation using in gel zymogram analysis combined with
mass spectroscopy-based peptide analysis of gut proteins
from larval Asian longhorned beetles, Anoplophora
glabripennis

Scott M. Geib1 , Ming Tien1 and Kelli Hoover2

Departments of 1 Biochemistry and Molecular Biology and 2 Entomology, The Pennsylvania State University, University Park, Pennsylvania,
USA

Abstract Enzyme activities toward lignocellulose substrates were analyzed in the gut of
larval Asian longhorned beetle (Anoplophora glabripennis). Total protein was extracted
from gut contents of wild collected larvae from an invasive population in Worchester,
MA, USA. From these protein extracts, lignocellulolytic activities were measured (β-1,4-
endoglucanase, β-1,4-glucosidase and birch wood xylanase). β-1,4-glucosidase activity
was 0.075 μmol glucose/mg protein per min, endoglucanase activity was measured at
0.41 μmol glucose/mg protein per min and xylanase activity was 0.058 μmol xylose/mg
protein per min. To identify specific enzymes that may provide these activities, zymo-
gram analysis was performed to detect enzymes active toward carboxymethyl cellulose
(CMC), 4-methylumbelliferyl-β-D-glucopyranoside and birch wood xylan. Three protein
bands were found to be active toward CMC, three displayed β-1,4-glucosidase, and one
displayed xylanase activity. Proteins from active bands from these zymograms were then
identified by in-gel trypsin digestions followed by peptide identification by matrix-assisted
laser desorption ionization – time of flight – time of flight mass spectrometry (MS). A
custom A. glabripennis transcriptome database was used for peptide identification, giv-
ing highly significant matches in all MS analyses. These matches were then searched
against the National Center for Biotechnology Information database to provide annotation
to the transcripts and provide possible classification. From these analyses, we were able
to detect enzymes active toward cellulose and xylan, and proteins putatively involved in
lignocellulose degradation in the gut of this wood-feeding insect. Future research will be
focused on characterizing these enzymes through cloning and expression experiments and
understanding how the lignocellulose degradation system functions in the gut of this insect.
Key words beta-glucosidase, cellulase, Cerambycidae, endoglucanase, proteomics,
xylanase

Introduction

Correspondence: Scott M. Geib, 408 Althouse Lab-
oratory, Department of Biochemistry and Molecular
Biology, The Pennsylvania State University, University
Park, PA 16802, USA. Tel: 814 865 0658; fax: 814 863 7024;
email: smg283@psu.edu
The 30 × 30 Biofuels Initiative set by the US Depart-
ment of Energy is to replace 30% of the current gasoline
consumption with biofuels by 2030. It is expected that
a major contributor to biofuel stock will be cellulosic
ethanol. While cellulose represents the largest source of


C 2010 The Authors 253
Journal compilation 
C Institute of Zoology, Chinese Academy of Sciences
254 S. M. Geib et al.
renewable carbon on earth, accessibility to the sugar com-
ponent required to produce ethanol is limited by the high
costs associated with extracting (depolymerizing) sugars
from a complex lignocellulose substrate that is heavily
cross-linked and crystalline. This study aims to identify
enzymes produced in the gut of a xylophagous insect, the
Asian longhorned beetle (Anoplophora glabripennis) that
are involved in lignocellulose degradation. Mining this
environment for such enzymes can potentially yield new
enzymes for processing lignocellulolytic material into cel-
lulosic ethanol. Little is known about how wood-feeding
beetles, specifically longhorned beetles (Family: Ceram-
bycidae), degrade lignocellulose. Thus, the potential for
discovery of novel enzymes from these species is con-
siderable. Larval A. glabripennis grow and develop on
the inner-wood of living hardwood tree species. This in-
sect is unusual because it feeds in a wide diversity of
species of both healthy and weakened (dead/dying) trees
(Hanks, 1999). The beetles’ main food source contains
fully polymerized, cross-linked lignocellulose. Lignocel-
lulose degradation was hypothesized to be carried out,
at least in part, by gut symbionts. We found that the
A. glabripennis gut harbors a wide diversity of microbes,
many of which may produce enzymes capable of degrad-
ing lignocellulose (Geib et al., 2008, 2009).
Wood is composed of three polymeric materials:
cellulose, hemicellulose and lignin. Cellulose is a linear
polymer of glucose linked by β-1,4 glycosidic bonds, ac-
counting for approximately 45% of wood by weight. Its
linear structure and extensive hydrogen bonding increases
crystallinity of this macromolecule and decreases the ac-
cessibility of hydrolytic enzymes. Hemicellulose accounts
for approximately 25% of wood by weight and is also
linked by β-1,4 linkages. Unlike cellulose, hemicellulose
has much greater structural heterogeneity, containing sug-
ars (the most predominate being xylose), sugar acids and
acetyl esters as side groups from the linear polymer. These
side groups prevent efficient packing of the hemicel-
lulose fibrils and render hemicelluloses non-crystalline.
Lignin imparts structural rigidity in woody biomass with
phenylpropanoid units being the precursors of lignin (van
Rensburg et al., 2000). Oxidation of these phenols yields
free radicals, which undergo radical coupling to form a
polymer linked by over 12 types of chemical bonds. It
is the random nature of lignin cross-linking and its con-
densed and insoluble properties that makes lignin resistant
to most forms of microbial attack.
Cellulose digestion occurs widely in different taxa of
insects (Martin, 1983) and the proportion of ingested cel-
lulose digested can be extremely high (up to 99%) (Prins
& Kreulen, 1991). Cellulolytic enzymes may originate
from gut symbionts, ingestion of enzymes produced by
Journal compilation 
C Institute of Zoology, Chinese Academy of Sciences, Insect Science, 17, 253–264
wood decay fungi, the insect itself, or some combination
of these (Martin, 1983; Breznak & Brune, 1994; Watanabe
& Tokuda, 2001; Brune, 2003; Suh et al., 2005). Over-
all, very little is known about how cellulose is digested
in insects, with the exception of termites or other insects
living in decayed wood. Some longhorned beetles make
their own endoglucanases and β-glucosidases, but to-date
no insects have been found to produce exoglucanases
(Sugimura et al., 2003; Lee et al., 2004).
It is well known that the digestibility of cellulose de-
creases as the lignin content of plant biomass increases
(Hatfield et al., 1999). Some wood-eating insects are able
to overcome the lignin barrier, and obtain help with cel-
lulose digestion by feeding on wood that was previously
degraded by environmental fungi (Kukor et al., 1988). In
fact, in a group of higher termites (Termitidae, Macroter-
mitidae), the colony actually cultivates a basidiomycete
fungus within the nest (Hyodo et al., 2000; Taprab et al.,
2005). Basidiomycetes are known to cause white-rot de-
cay in wood and can produce lignin and Mn peroxidases,
as well as laccases, which degrade and depolymerize the
lignin macromolecule (Taprab et al., 2005). This precon-
ditioning permits greater access to cellulose. Other insects
feed on dead, rotting wood colonized with white or brown
rot fungi. It was previously thought that substantial lignin
degradation did not occur within the gut of wood-feeding
insects (Ohkuma, 2003). However, recently we reported
that lignin is modified during passage of wood through the
A. glabripennis gut, as well as the gut of the Pacific damp-
wood termite (Geib et al., 2008). While the biochemical
process of lignin degradation was partially characterized,
we do not know what enzymes are involved in overcom-
ing the lignin barrier in A. glabripennis. Herein, we ex-
amine the lignocellulolytic ability of larval A. glabripen-
nis. Enzyme assays were performed on total gut enzyme
extracts. In addition, zymogram analyses toward hy-
drolysis of different lignocellulose components were
performed along with in-gel trypsin digestion/peptide
sequencing to identify specific enzymes involved in wood
degradation in this insect gut.
Materials and methods
Insect collection
Anoplophora glabripennis were field collected in con-
junction with eradication efforts by the US Department
of Agriculture Animal and Plant Health Inspection Ser-
vice Plant Protection and Quarantine (USDA-APHIS-
PPQ). Infested trees were located by the presence of
exit holes and dieback of the trees. Several trees were

C 2010 The Authors

cut for this study in January 2009, from a single small
stand of mature silver maple (Acer saccharinum) on
a property in urban Worcester, MA, US (42◦ 18 18 N,
71◦ 48 00 W). Trees were cut into short (0.5–1.0 meter)
bolts and transported to the Pennsylvania State University
Quarantine Research Lab following our USDA permit
guidelines. Due to the time of year that the wood was
collected, the larvae were not actively feeding so the
bolts were stored in an aluminum screen cage within our
quarantine facility and maintained at room temperature
(≈22◦ C) for 2 months to allow the insects to resume feed-
ing when collected for dissection, as evidenced by frass
(feces) production from the bolts. At this point the bolts
were split and late instar larva were removed for dissec-
tion. Larvae were immediately put on ice after removal
from trees and dissected.
Anoplophora glabripennis larval gut dissection
and protein extraction
Larvae removed from trees were immediately chilled
and dissected within 1 h in a laminar flow hood to maintain
sterility using sterile dissection tools. Before dissection,
larvae were surface-sterilized in 70% ethanol for 1 min
and rinsed in sterile water. To remove the entire, intact
gut, the cuticle was cut laterally and the gut was ligated
at either end to isolate an intact gut section between the
anterior midgut and posterior hindgut. This portion was
then carefully transferred to a new sterile dissection dish.
The gut contents were then removed by carefully cut-
ting the gut open and transferring the contents into a mi-
crofuge tube containing 0.05 mol/L sodium citrate buffer,
pH 5.5. Gut contents from 15 larvae were combined to
obtain sufficient protein. The sample was then homog-
enized with a micropestel, centrifuged at 12 000 g for
5 min, and the supernatant was transferred to a new tube.
This supernatant was used as gut enzyme extract. Three
replicate samples were created in this manner.
Cellulase and xylanase assays
Activities of β-1,4-glucosidase, β-1,4-endoglucanase
and β-1,4-xylanase were measured from A. glabripen-
nis gut content enzyme extracts incubated with cellu-
lose or xylan substrates based on the release of re-
ducing sugars measured by the dinitrosalicylic acid
(DNS) assay (Bernfeld, 1955; Miller, 1959). Each assay
was performed in triplicate. Protein concentration was
measured using the Bradford method (Bradford, 1976;
Bollag et al., 1996) using bovine serum albumin as a
protein standard (0–20 μg) and samples were diluted

C 2010 The Authors
Journal compilation 
C Institute of Zoology, Chinese Academy of Sciences, Insect Science, 17, 253–264
Proteomic analysis of ALB gut cellulases 255
to a working concentration of approximately 40 μg/mL
in sodium citrate buffer (50 mmol/L, pH 5.5). For
β-glucosidase activity, 750 μL of a 2% salicin solution (in
50 mmol/L sodium citrate buffer, pH 5.5) was combined
with 30 μg (750 μL of 40 μg/mL dilution) of gut enzyme
extract. For β-endoglucanase activity 750 μL of a 2%
carboxymethyl cellulose (CMC) solution (Kukor et al.,
1988) (in 50 mmol/L sodium citrate buffer, pH 5.5) was
combined with 750 μL of gut content enzyme extract.
For xylanase activity, 750 μL of a 1% xylan solution (in
50 mmol/L sodium citrate buffer, pH 5.5, xylan from birch
wood, Sigma-Aldrich, St Louis, MO, US) was combined
with 30 μg (750 μL of 40 μg/mL dilution) of enzyme ex-
tract. For all assays, background sugars were subtracted
from the end product by removing 100 μL from the re-
action mixture and measuring sugar content at time 0.
Reaction mixtures were incubated at 37◦ C with 100 μL
aliquots removed after 30, 60, 90, 120 and 240 min. For
each aliquot, 100 μL DNS reagent were added to halt
enzyme activity (Miller, 1959). Samples were then incu-
bated in a boiling water bath for 8 min and absorbance of a
150 μL aliquot was read at 540 nm on a SpectraMax(tm)
190 microplate reader (Molecular Devices Corp.,
Sunndale, CA, US) along with glucose standards (stan-
dard curve, 20–1000 μg).
Zymogram analysis
Sodium dodecyl sulfate – polyacrylamide gel elec-
trophoresis (SDS-PAGE) or native PAGE gels (Laemmli,
1970) were performed with some alterations to detect ac-
tivity of cellulases, β-glucosidase or xylanases through
zymogram techniques (Schwarz et al., 1987; Painbeni
et al., 1992; Her et al., 1999; Chavez et al., 2002). For
the CMC zymogram, a 12% SDS-PAGE separation gel
was poured containing 0.1% carboxymethyl cellulose. A.
glabripennis gut enzyme extracts were loaded onto half
of the gel at three different amounts of protein (7, 20 and
40 μg protein/lane). This loading pattern was repeated on
the other half of the gel so that the gel could be cut verti-
cally in half after electrophoresis to produce two identical
acrylamide gels. The first half of the gel was stained with
colloidal blue stain to visualize proteins. The second half
was used for zymogram analysis. For zymogram analysis,
the gel was rinsed in sodium citrate buffer (50 mmol/L,
pH 5.5) containing 1% Triton X-100 for 1 h at room
temperature to remove SDS (Her et al., 1999). This was
followed by incubation for 1.5 h in sodium citrate buffer
(50 mmol/L, pH 5.5) to allow for enzyme activity against
the substrate. Then the gel was stained with 0.1% Congo
red for 30 min and destained in 1 mol/L NaCl to reveal
256 S. M. Geib et al.
zones of clearing. The gel was imaged under ultraviolet
light and aligned with colloidal blue stained gels.
For β-glucosidase and xylanase zymograms, we used
native PAGE to maintain protein activity during elec-
trophoresis. Twelve percent native PAGE gels (Laemmli,
1970) were created lacking SDS, and contained either
2 mmol/L 4-methylumbelliferyl-β-D-glucopyranoside, or
0.1% birch wood xylan for the β-glucosidase or xylanase
zymograms, respectively (Schwarz et al., 1987; Painbeni
et al., 1992; Her et al., 1999; Chavez et al., 2002). A.
glabripennis gut enzyme extracts were loaded onto half
of the gels at three different amounts of protein (7, 20
and 40 μg protein/lane) as described above with half
for protein staining and the other half for zymograms.
Electrophoresis was carried out in standard Tris-glycine
buffer (25 mmol/L Tris, 192 mmol/L glycine, pH 8.8)
lacking SDS and the gel was maintained at 4◦ C dur-
ing electrophoresis. Zymogram analysis was performed
by incubating the gels for 1.5 h in sodium citrate buffer
(50 mmol/L, pH 5.5) to allow for enzyme activity against
the substrates. At this point, the β-glucosidase zymogram
gel was visualized under ultraviolet light to detect fluores-
cence of 4-methylumbelliferone from the substrate where
β-glucosidase activity was present. Xylanase activity was
visualized by first staining the gel with 0.1% Congo red
for 30 min and destaining in 1 mol/L NaCl to reveal zones
of clearing.
Trypsin digestion of selected protein bands
and MALDI-TOF-TOF analysis
Bands correlating to activity based on zymogram anal-
ysis were excised from reference gels and prepared
for matrix-assisted laser desorption ionization – time
of flight – time of flight (MALDI-TOF-TOF) analy-
sis by trypsin digestion. Bands of interest (labeled in
Figs. 2–4) were excised using a clean razor blade and
cut into 1 mm2 pieces. Gel pieces were destained with
200 μL of 100 mmol/L ammonium bicarbonate (pH 8.0)
in 50% acetonitrile, with two repeated 45 min washes at
37◦ C. After destaining, gel pieces were dried in a Speed-
Vac (Thermo Scientific, Waltham, MA, US), reduced in
10 mmol/L dithiothreitol in 25 mmol/L ammonium bi-
carbonate (pH 8.0) for 15 min at 37◦ C, and alkylated in
20 mmol/L iodoacetamide in 25 mmol/L ammonium bi-
carbonate (pH 8.0) for 45 min at 37◦ C in the dark. Three
washes with 25 mmol/L ammonium bicarbonate were
then performed to remove iodacetamide. The gel pieces
were dried again and then rehydrated in 0.02 μg/μL of
Trypsin Gold, MS grade (Promega Corporation, Madison,
WI, US) in 10% acetonitrile, 40 mmol/L ammonium bi-
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C Institute of Zoology, Chinese Academy of Sciences, Insect Science, 17, 253–264
carbonate (pH 8.0) and 0.1% w/v n-octylglucoside. Di-
gestion was performed at 37◦ C for 18 h. After digestion,
peptides were extracted in 50% acetonitrile, 0.1% w/v n-
octylglucoside and dried in a SpeedVac. The dried sample
was then rinsed three times with 100 μL of water, allowing
the samples to dry completely between each wash. After
the final wash, the peptides were resuspended in water.
Samples were then concentrated and cleaned for mass
spectroscopy using a ZipTip SCX pipette tip (Millipore
Corporation, Billerica, MA, US).
Samples were analyzed at the Penn State Hershey Med-
ical Center Mass Spectrometry Core Research Facility
on an Applied Biosystems 4800 Proteomic Analyzer
(MALDI-TOF-TOF). For each sample, the molecular
mass of each peptide fragment was measured, and then the
amino acid sequence of the top 10 most abundant peptides
was determined using ProteinPilot 3.0 software coupled
with the MALDI-TOF-TOF analyzer (Applied Biosys-
tems, Foster City, CA, US). Within ProteinPilot, proteins
were identified by searching a custom A. glabripennis
gut transcriptome database consisting of 5 959 assembled
contigs (average contig size ∼900 bp) from a cDNA li-
brary sequenced using a 454 FLX pyrosequencer (Roche
Technologies, Branford, CT, US). The transcriptome li-
brary was translated into all possible amino acid se-
quences across all six frames, and this resulting amino
acid database was used in the ProteinPilot software.
In addition, the Tribolium castaneum predicted protein
database (obtained from http://beetlebase.org), as well as
the termite gut metagenome, Fusarium solani, and Phane-
rochaete chrysosporium predicted protein databases (ob-
tained from http://genome.jgi-psf.org) were used, but ei-
ther no significant matches were obtained from these
databases or a higher significance score was obtained
when using our custom ALB (Asian longhorned bee-
tle) database. ProteinPilot identifies proteins from double
mass spectroscopy (MS/MS) spectra using the Paragon
Algorithm, with an “unused” score higher than 1.3 rep-
resenting a significant protein match (at 95% confi-
dence). For each sample, significant protein matches
to the transcriptome database were identified using this
method, and the identified contigs from the transcrip-
tome database were searched against the National Center
for Biotechnology Information (NCBI) nr database and
annotated.
Results
Enzyme activity of A. glabripennis gut contents
Activities of several lignocellulolytic enzymes were
assayed from gut extracts of larval A. glabripennis.

C 2010 The Authors
 Proteomic analysis of ALB gut cellulases 257

β-1,4-glucosidase activity was 0.075 μmol glucose/mg

protein per min based on the release of β-1,4-linked glu-
cose from salicin (Fig. 1A). Endoglucanase activity, de-
termined by reducing sugar release from carboxymethyl
cellulose, was 0.41 μmol glucose/mg protein per min
(Fig. 1B) and xylanase activity, determined by the release
of reducing sugars from birch wood xylan was 0.058 μmol
xylose/mg protein per min (Fig. 1C).

Zymogram analyses

Zymogram analysis for cellulase activity as mea-

sured by degradation of carboxymethyl cellulose showed
that at least three enzymes are responsible for the
measured cellulase activity. Three major zones of
clearing were observed on the zymogram gel, with sizes
of approximately 28, 30 and 40 kDa (Fig. 2, lane C). Ex-
amination of the colloidal blue-stained SDS-PAGE gel
showed protein bands corresponding with these zones
of clearing, with a major band at 28 kDa, a less abun-
dant band at 40 kDa, and a faint band at 30 kDa
(Fig. 2, lane B). β-glucosidase zymogram analysis
revealed two active bands at approximately 38 and 60 kDa.
The birch wood xylan zymogram analysis revealed a sin-
gle band of activity at approximately 34 kDa (Figs. 3
and 4).

MALDI-TOF-TOF identification of proteins

from active bands

Bands corresponding to activity on the endoglu-

canase, β-glucosidase and xylanase zymograms were
excised for peptide analysis (denoted with arrows in
Figs. 2–4). MALDI-TOF-TOF analysis run on the
Applied Biosystems 4800 Analyzer identifies proteins in
a sample by comparing the peptide sequence obtained
from the analysis to a database. The significance of a
match is calculated by the ProteinPilot software with sig-
nificant scores having an “unused” value greater than 1.3.
Table 1 presents the significant matches for each gel band
analyzed, with the percentage coverage of the peptides
identified against the matching protein in the database,
and the number of peptides that matched the protein at
95% confidence. In addition, the contig length from the
transcriptome database, as well as the number of 454 FLX
reads that were assembled to create that contig, are listed Fig. 1 Cellulase and xylanase activity of A. glabripennis gut
along with a protein description based on the top NCBI nr enzyme extract. Enzyme activity of (A) beta-glucosidase, (B)
BLAST match and various BLAST statistics (Table 1). endoglucanase, and (C) xylanase, measured by release of reduc-
Also, the corresponding amino acid sequence of each ing sugar from each substrate by 30 μg of total gut protein over
identified protein from the database is listed in FASTA 180 min. One unit (U) of activity is the amount of enzyme to
format (supplementary information). hydrolyze 1 μmol of reducing sugar per minute.


C 2010 The Authors
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C Institute of Zoology, Chinese Academy of Sciences, Insect Science, 17, 253–264
258 S. M. Geib et al.
Fig. 2 Carboxymethyl cellulose (CMC) zymogram of
A. glabripennis larval gut contents. Electrophoresis of 20 μg of
A. glabripennis gut contents. Protein standard is on the left (lane
A), with band sizes labeled (kDa). Lane B is a colloidal blue-
stained sample and lane C is the corresponding CMC zymogram
stained with Congo red. Dark bands represent zones of clear-
ing (under ultraviolet light). Three major zones of clearing are
present at approximately 28, 30 and 40 kDa and matched to
protein bands on the colloidal blue-stained lane. Arrows de-
pict corresponding protein bands that were excised for matrix-
assisted laser desorption ionization – time of flight – time of
flight analysis; band name with protein identification is listed in
Table 1.
For the CMC zymogram, none of the three bands on the
gel gave protein matches that could be clearly identified as
cellulases. The first band was identified as a “thaumatin-
like” protein, the second as a “serine proteinase,” and
the third consisted of three proteins that either had no
significant match in the NCBI database or matched to an
unknown protein with no annotation.
For the β-glucosidase gel bands, both pro-
duced matches to glycoside hydrolases, including
β-glucosidases along with other proteins. The band ex-
cised from the xylanase zymogram analysis also produced
a match to a glycoside hydrolase, as well as a sucrose-6-
phosphate hydrolase.
Journal compilation 
C Institute of Zoology, Chinese Academy of Sciences, Insect Science, 17, 253–264
Fig. 3 Beta-glucosidase zymogram of A. glabripennis gut
contents protein extract using native polyacrylamide gel ele-
crophoresis. Electrophoresis of 30 μg of A. glabripennis gut
contents. Protein standard is on the left (lane A), with band
sizes labeled (kDa). Lane B is a colloidal blue-stained sample
and lane C is the corresponding beta-glucosidase zymogram
visualized under ultraviolet (UV) light. Dark bands represent
zones of clearing (under UV light). Two major zones of clear-
ing are present at approximately 38 and 60 kDa and matched
to protein bands on the colloidal blue-stained lane. Arrows de-
pict corresponding protein bands that were excised for matrix-
assisted laser desorption ionization – time of flight – time of
flight analysis; band name with protein identification is listed in
Table 1.
Discussion
In this study, we characterized the enzymes involved in
lignocellulose degradation found in the larval stages of
A. glabripennis. Figure 1 presents the enzyme activity
of the Asian longhorned beetle larval gut contents toward
β-1,4-endoglucanase, β-1,4-glucosidase and xylanase ac-
tivity. The enzyme activities measured here in the Asian
longhorned beetle are similar to those seen in termites
and other cerambycid beetle species (Kukor et al., 1988;
Tokuda et al., 2005; Smith et al., 2009).
Zymograms were performed to assess the number of
enzymes responsible for the observed activities. The

C2010 The Authors

Fig. 4 Birch wood xylan zymogram of A. glabripennis gut
contents protein extract using native polyacrylamide gel ele-
crophoresis. Electrophoresis of 30 μg of A. glabripennis gut
contents. Protein standard is on the left (lane A), with band
sizes labeled (kDa). Lane B is a colloidal blue-stained sample
and lane C is the corresponding birch wood xylan zymogram
stained with Congo red. Dark bands represent zones of clearing
(under ultraviolet light). One major zone of clearing is present
at approximately 34 kDa and can be matched to a protein band
on the colloidal blue-stained lane. An arrow depicts the corre-
sponding protein band that was excised for matrix-assisted laser
desorption ionization – time of flight – time of flight analysis;
band name with protein identification is listed in Table 1.
zymograms using carboxymethyl cellulose as a sub-
strate showed several active bands, two at approximately
28–30 kDa, and one slightly larger than 36 kDa. This
is consistent with several enzymes present in this gut
system that are able to act upon non-crystalline cel-
lulose and liberate reducing sugars. The zymograms
were of gels separating the proteins only in one di-
mension. The Coomassie-blue staining of similar gels
showed that while the activity bands did not re-
flect pure proteins, there was still a possibility that
they could be identified by MALDI-TOF-TOF. Bands
CMC-1, CMC-2 and CMC-3 did not yield any peptides
that could be clearly identified to correspond to a cellu-
lase gene. For CMC-1, a single protein type was identified
as thaumatin-like proteins, which we would not expect to
exhibit hydrolase activity. From band CMC-2 a single
proteinase was identified, which is again not likely to

C 2010 The Authors
Journal compilation 
C Institute of Zoology, Chinese Academy of Sciences, Insect Science, 17, 253–264
Proteomic analysis of ALB gut cellulases 259
be involved in cellulose degradation. CMC-3 identified
three unknown proteins. One, contigE00782, had a sig-
nificant match to a T. castaneum protein, NCBI accession
number XP_975796, but the function of this protein is not
known. The other two proteins matched to contigs E00961
and E04148, which had no significant matches to any se-
quence in the NCBI database, suggesting that these are
previously unknown proteins. While it is possible that one
or all of these proteins are involved in cellulose hydrolysis,
the more likely explanation is that the proteins of inter-
est in the CMC bands are not the most abundant proteins
from the fragments excised. Further separation of these
proteins, for example through two-dimensional gel elec-
trophoresis, may yield more pure protein spots, permit-
ting identification of specific cellulase enzymes. Our tran-
scriptome database has several contigs that match to puta-
tive cellulase genes. Ideally, we would like to demonstrate
their cellulolytic activity through zymogram analysis to
confirm the annotation. In other cerambycid species, sev-
eral families of glycoside hydrolases have been described
that have endoglucanase activity. In the mulberry longi-
corn beetle, Apriona germari, three insect endoglucanase
genes have been identified, two classified in glycoside
hydrolase family 45 and one in family 5 (Lee et al., 2004,
2005). In the yellow-spotted longicorn beetle, Psacothea
hilaris, an endogenous family 5 endoglucanase has been
characterized (Sugimura et al., 2003).
Activity was also observed with zymograms for β-
glucosidase and birch wood xylanase. Unlike the zymo-
grams containing SDS described above, the molecular
weight obtained from the native gels used here may not
accurately reflect the actual mass of the proteins since
proteins do not migrate solely based on mass under native
conditions (Bollag et al., 1996). From the β-glucosidase
zymogram, three fragments were excised from the pro-
tein gel for MALDI-MS-MS, which corresponded with
the two active zones on the zymogram. Peptide analy-
sis of the first band, BG-1, gave rise to six peptides that
matched to the transcriptome dataset giving a significant
score (“unused” > 1.3), matching to four different contigs
within this dataset. Interestingly, all four of these contigs
had the highest BLAST match to the same gene from Tri-
bolium castaneum, NCBI accession number XP_972437.
This gene is a putative glycoside hydrolase family 1 β-
glucosidase based on conserved domains. However, all
four contigs from the ALB transcriptome were distinct,
suggesting that there may be multiple hydrolase genes
present that perform similar functions.
It was not clear which protein band matched the
second active zone due to a wide band of activity
(Fig. 3), so two bands were excised, BG-2 and BG-
3. Analysis of the second band, BG-2, gave rise to 10
 260

Table 1 Proteins identified from in gel digestion/MALDI-TOF-TOF analysis of active bands of zymogram gels.
Protein % coverage # peptides Transcriptome #
Gel number Unused % (95% matched contig Contig reads Sequence Hit (NCBI Alignment
Organism E-value Similarity Score Positives
band from score coverage confidence) (95% matched length in description accession #) length
band confidence) contig
S. M. Geib et al.

CMC-1 1 2.56 36.6 14.1 1 contigE04500 213 18 Thaumatin-like Tribolium XP_975175 3.942E-15 64 83.96 65 42
protein castaneum
2 2.28 17.5 10.9 1 contigE00172 434 49 Thaumatin-like Tribolium XP_975175 1.273E-29 92 132.11 67 62
protein castaneum
3 2 32.9 12.5 1 contigE04351 266 33 Thaumatin-like Tribolium XP_968724 2.423E-28 80 127.87 87 70
protein castaneum
CMC-2 1 5.05 7.8 5.3 2 contigE00651 846 24 Chymotrypsin-like Costelytra ABZ04021 2.574E-43 60 179.49 264 159
serine proteinase zealandica
CMC-3 1 6 22.5 22.5 5 contigE00782 740 13 Unknown protein Tribolium XP_975796 8.391E-10 51 67.78 209 107

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castaneum

C

2 3.05 8.8 4.3 1 contigE00961 1147 77 No BLAST match
3 2 4.5 4.5 1 contigE04148 1153 96 No BLAST match
BG-1 1 2.85 7.1 4.0 2 contigP00192 768 10 Beta-glucosidase Tribolium XP_972437 8.127E-80 74 252.68 129 96
castaneum
2 2.01 4.1 2.3 2 contigE00211 1308 71 Glycoside hydrolases Tribolium XP_972437 1.47E-143 73 513.46 429 315
castaneum
3 2 2.7 2.7 1 contigE04507 1096 118 Beta-glucosidase Tribolium XP_972437 5.71E-111 72 404.83 353 255
castaneum
4 1.4 3.4 1.5 1 contigE03980 1600 186 Glycoside hydrolases Tribolium XP_972437 3.71E-171 74 605.52 499 370
castaneum
BG-2 1 8 5.9 5.9 4 contigE00216 2492 176 Neutral Tribolium XP_967103 0 63 677.17 735 464
alpha-glucosidase castaneum
ab
2 4 6.2 4.0 2 contigP00027 1505 101 Carboxylesterase Tribolium XP_972251 1.73E-90 59 337.42 495 296
castaneum
3 2.06 7.1 4.5 1 contigE04148 1153 96 No BLAST match
4 2 5.3 5.3 1 contigE00651 846 24 Chymotrypsin-like Costelytra ABZ04021 2.574E-43 60 179.49 264 159
serine proteinase zealandica
5 1.4 2.7 2.7 2 contigE03986 2692 93 Neutral Tribolium XP_967022 0 66 463.00 408 270
alpha-glucosidase castaneum
ab
BG-3 1 8 19.1 19.1 4 contigE00961 1147 77 No BLAST match
2 3.3 10.0 10.0 2 contigE00651 846 24 Chymotrypsin-like Costelytra ABZ04021 2.574E-43 60 179.49 264 159

C
serine proteinase zealandica


3 2 4.4 1.5 1 contigE04156 1556 203 Neutral Tribolium XP_967103 5.15E-170 70 601.67 521 368
alpha-glucosidase castaneum
ab

Continued

Institute of Zoology, Chinese Academy of Sciences, Insect Science, 17, 253–264

2010 The Authors
 C

Table 1 Continued
Protein % coverage # peptides Transcriptome #
Gel number Unused % (95% matched contig Contig reads Sequence Hit (NCBI Alignment
Organism E-value Similarity Score Positives
band from score coverage confidence) (95% matched length in description accession #) length

2010 The Authors

band confidence) contig

Journal compilation 
4 2 5.3 2.0 1 contigE04111 1198 261 Carboxylesterase Tribolium XP_970253 3.263E-70 60 269.63 383 231
castaneum
5 2 1.9 1.9 1 contigP00021 1585 49 Carboxylesterase Tribolium XP_970253 1.67E-123 64 447.20 506 326
castaneum
X-1 1 4.19 9.2 6.1 2 contigP00237 1261 25 Sucrose-6-phosphate Streptococcus ZP_01817526 1.34E-100 70 321.63 168 119
hydrolase pneumoniae
SP3-BS71
2 2 6.3 6.3 1 contigP01396 475 20 Glycoside hydrolases Tribolium XP_972437 5.323E-52 79 206.45 159 127
castaneum
H-1 1 5.7 10.7 8.8 3 contigP01221 1125 24 Gram negative XP_970010 4.95E-142 76 508.06 368 281
bacteria binding
protein 1
2 4 14.3 14.3 3 contigE00782 740 13 Unknown protein Tribolium XP_975796 8.391E-10 51 67.78 209 107
castaneum
3 3.87 5.0 2.7 2 contigE00144 2635 85 Neutral Tribolium XP_967022 0 68 704.90 427 294
alpha-glucosidase castaneum
ab
4 2 12.5 12.5 1 contigP00184 241 2 Neutral Tribolium XP_967103 1.51E-14 68 82.03 80 55
alpha-glucosidase castaneum
ab
H-2 1 3.55 6.4 4.2 2 contigE00151 1523 303 Plasma Tribolium XP_971511 0 80 639.80 432 348
alpha-l-fucosidase castaneum
H-3 1 5.07 5.9 4.0 2 contigE00144 2635 85 Neutral Tribolium XP_967022 0 68 704.90 427 294
alpha-glucosidase castaneum
ab
2 2 8.6 8.6 1 contigP01299 492 17 Chitinase 3 Monochamus BAF49605 1.403E-47 75 191.82 137 103
alternatus
3 2 2.1 2.1 1 contigP01221 1125 24 Gram-negative XP_970010 4.95E-142 76 508.06 368 281
bacteria binding
protein 1

C Institute of Zoology, Chinese Academy of Sciences, Insect Science, 17, 253–264

4 2 12.5 12.5 1 contigP00184 241 2 Neutral Tribolium XP_967103 1.51E-14 68 82.03 80 55
alpha-glucosidase castaneum
ab
5 1.7 8.4 8.4 1 contigP01217 467 4 No BLAST match
6 1.3 4.4 2.9 1 contigE04156 1556 203 Neutral Tribolium XP_967103 5.15E-170 70 601.67 521 368
alpha-glucosidase castaneum
ab

BLAST, basic local alignment search tool.

Proteomic analysis of ALB gut cellulases
261
262 S. M. Geib et al.
peptides that matched to five contigs in the dataset.
Of these, contigE00216 had the highest score (8.0)
with four of the peptides matching to this contig.
Searching this contig against NCBI gave a match to a
glycoside hydrolase family 31 α-glucosidase from T. cas-
taneum, NCBI accession number XP_967103. In addi-
tion, a second contig, E03986 matched to another family
31 α-glucosidase, NCBI accession number XP_967022.
Analysis of protein band BG-3 identified contigE04156
matching to the same T. castaneum α-glucosidase as
the first protein identified in BG-2. These findings sug-
gest that there are at least three β-glucosidases in the A.
glabripennis gut that appear to have activity toward β-1,4
linkages. Known β-glucosidases fall into three glycoside
hydrolase families, GH 1, 3 and 9, but animal-derived
β-glucosidases have only been identified in families 1
and 9. In other coleopteran species, the number of β-
glucosidases identified to date have been relatively large.
For example, two β-glucosidases were identified from Er-
gates faber with sizes of 57 and 70 kDa (Chararas et al.,
1983). In Tenebrio molitor, two β-glucosidase genes were
identified, βGly1 and βGly2, which fall into GH family
1 (Ferreira et al., 2001).
Only one single insect-derived xylanase gene has been
cloned. It encodes a 75 kDa protein in GH family 11
derived from Phaedon cochleariae (Girard & Jouanin,
1999). Several insect-associated microbes have been
shown to produce xylanases used by insects, which in-
clude but are not limited to protists in termite gut systems
(Arakawa et al., 2009), bacteria associated with beetles
(Zhou et al., 2009), and fungal species associated with
siricids (Kukor & Martin, 1983). Analysis of the pro-
tein band that matched to hydrolysis of birch wood xylan
identified two contigs. Of these, contig P01396 matched
to a glycoside hydrolase in T. castaneum, NCBI accession
number XP_972437. This is the same gene that matched
to the proteins in band BG-1, but the match in this case
had a lower score. While the database suggests that this
is a β-glucosidase, our zymogram analysis suggests that
the activity is directed toward β-1,4-xylan.
We demonstrated here that larval Asian longhorned
beetles have enzymes present in their gut that aid in
the degradation of both xylan and cellulose. Zymo-
gram analysis was used to visualized specific activi-
ties of these enzymes; coupled with tandem mass spec-
trometry analysis of peptide digestions we were able to
identify specific novel proteins involved in wood degra-
dation in this system. These results will lead to future
studies, where these targeted proteins that have lignocel-
lulose activity will be cloned, expressed and characterized
as done previously in other insect and invertebrate sys-
tems (Smant, 1998; Sugimura et al., 2003; Suzuki et al.,
Journal compilation 
C Institute of Zoology, Chinese Academy of Sciences, Insect Science, 17, 253–264
2003; Lee et al., 2004, 2005). Expression and charac-
terization of specific enzymes will allow for comparison
of enzyme activity and substrate specificity compared to
other known insect cellulases/xylanases, as well as mi-
crobial and fungal-derived enzymes. In addition, analysis
of combinations of enzymes, for example cellulases and
beta-glucosidases together, to identify the complexes that
are most efficient in conversion of lignocellulose are of
interest. Together, these future studies will lead toward a
better understanding of how insect-derived enzymes can
be utilized in industrial lignocellulose processing and if
there are advantages over microbial systems.
Acknowledgments
We thank A. Sawyer’s group at USDA APHIS and
K. Gooch at the Massachusetts Department of Con-
servation and Recreation for assistance collecting bee-
tles from Worcester, MA and A. and B. Stanley at the
Penn State Hershey Medical Center Mass Spectrometry
Core Research Facility for mass spectrometry analysis.
Funding for this project was provided by USDA-NRI-
CRSEES 2008-35504-04464, USDA-NRI-CREES 2009-
35302-05286, and the Alphawood Foundation, Chicago,
IL.
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Accepted December 15, 2009

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 Insect Science (2010) 17, 265–276, DOI 10.1111/j.1744-7917.2010.01327.x

ORIGINAL ARTICLE

Comparison of gut-associated and nest-associated microbial

communities of a fungus-growing termite (Odontotermes
yunnanensis)

Yan-Hua Long1,2 , Lei Xie2 , Ning Liu2 , Xing Yan2 , Ming-Hui Li2 , Mei-Zhen Fan1 and Qian Wang2
1
Provincial Key Laboratory of Microbial Pest Control, Anhui Agricultural University, Hefei, 2 Institute of Plant Physiology and Ecology,
Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai, China

Abstract The digestion of cellulose by fungus-growing termites involves a complex of

different organisms, such as the termites themselves, fungi and bacteria. To further investi-
gate the symbiotic relationships of fungus-growing termites, the microbial communities of
the termite gut and fungus combs of Odontotermes yunnanensis were examined. The ma-
jor fungus species was identified as Termitomyces sp. To compare the micro-organism
diversity between the digestive tract of termites and fungus combs, four polymerase
chain reaction clone libraries were created (two fungus-targeted internal transcribed spacer
[ITS] – ribosomal DNA [rDNA] libraries and two bacteria-targeted 16S rDNA libraries),
and one library of each type was produced for the host termite gut and the symbiotic fungus
comb. Results of the fungal clone libraries revealed that only Termitomyces sp. was detected
on the fungus comb; no non-Termitomyces fungi were detected. Meanwhile, the same fun-
gus was also found in the termite gut. The bacterial clone libraries showed higher numbers
and greater diversity of bacteria in the termite gut than in the fungus comb. Both bacte-
rial clone libraries from the insect gut included Firmicutes, Bacteroidetes, Proteobacteria,
Spirochaetes, Nitrospira, Deferribacteres, and Fibrobacteres, whereas the bacterial clone
libraries from the fungal comb only contained Firmicutes, Bacteroidetes, Proteobacteria,
and Acidobacteris.
Key words fungus comb, gut, ITS, phylotype, termite, Termitomyces

Introduction ally classified at the taxonomic rank of the order Isoptera.

Termites are the major contributors to the biodegrada-
tion of lignocellulose and hemicellulose in tropical areas
(Lee & Wood, 1971). Therefore, these insects are im-
portant not only for their roles in carbon turnover in the
environment, but also as potential sources of biochemi-
cal catalysts for converting wood into biofuels (Warnecke
et al., 2007). Termites are a group of social insects usu-
Correspondence: Qian Wang, Institute of Plant Physiology
and Ecology, Shanghai Institutes for Biological Sciences, Chi-
nese Academy of Sciences, 300 Fenglin Road, Shanghai, 200032
China. Tel: +86 21 54924046; fax: +86 21 54920078; email:
wangqian@sippe.ac.cn
This group contains five families of lower termites and
one family of higher termites (Noirot, 1992). The special
subfamily of higher termites, Macrotermitinae, which can
digest plant litter with high efficiency, is also referred to
as fungus-growing termites. These termites have gained
the widespread interest of researchers due to their symbi-
otic relationship with basidiomycete fungi of the genus
Termitomyces (Agaricales: Tricholomataceae) (Abe &
Matsumoto, 1979; Wood & Sands, 1978).
Fungus-growing termites are distributed throughout
tropical and subtropical areas in Africa and Asia. They
have evolved into approximately 330 extant species,
belonging to approximately 12 genera (Kambhampati
& Eggleton, 2000). Inside the nests of these termites,
Termitomyces is cultured by termites on a special


C 2010 The Authors 265
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C Institute of Zoology, Chinese Academy of Sciences
266 Y. Long et al.
structure called the fungus comb. The comb is constructed
from undigested dead plant material and termite primary
feces; it is then inoculated with asexual spores of the
fungal symbiont (Leuthold et al., 1989). A few weeks af-
ter the inoculation, innumerable conidia rich in nitrogen
form many white nodules on the fungus comb. The host
termites feed on the mature parts of the fungus comb; the
mycelium then develops on the surface of the comb and
on the fungal nodules for nutrition (Lefèvre & Bignell,
2002). Fecal pellets (primary feces) produced by workers
(a caste of the termite) are added continuously to the top
of the comb, and fungal mycelium rapidly develops in the
newly added substrate.
Termites and gut bacteria, together with Termitomyces
fungi, form a symbiotic complex (Bignell, 2000); how-
ever, the relationship within the complex and the mech-
anism of cellulose digestion remain unclear. Several hy-
potheses concerning the main role of Termitomyces fungi
have been proposed. First, the fungi are presumed to
possess the ability to degrade lignin to make cellulose
more susceptible to attack by the termites’ own cellu-
lase (Hyodo et al., 2000; Johjima et al., 2003b; Taprab
et al., 2005). Veivers et al. (1991) supported this view and
found that symbiotic fungi in association with Macroter-
mes michaelseni and M. subhyalinus could not completely
degrade cellulose. Ohkuma (2003) also reviewed evidence
that higher termites relied primarily on endogenous cel-
lulases for cellulose digestion. Second, Martin & Martin
(1978) and Matoub & Rouland (1995) proposed that sym-
biotic fungi provide termites with cellulase and xylanases.
In an experiment on enzyme activity detection, Deng
et al. (2008) demonstrated that the symbiotic fungus had
a synergistic effect on the cellulase activity of Odontoter-
mes formosanus. Other studies (Sands, 1969; Darlington,
1994; Hyodo et al., 2000) have identified a number of fun-
gal cellulases, further supporting the results of Deng et al.
(2008). Third, the fungi are thought to serve as nitrogen
fixers, providing termites with nitrogen-rich food (Mat-
sumoto, 1976). Hyodo (2003) further explained that the
main role of the symbiotic fungus is to degrade lignin
in Macrotermes spp., but it does not serve as a food
source in Odontotermes spp., Hypotermes makbamen-
sis, Ancistrotermes pakistanicus, or Pseudacantbotermes
militaris.
Odontotermes yunnanensis, a species of fungus-
growing termite, is widely distributed throughout Yun-
nan Province, China, particularly in the Xishuang Banna
area. These termites play an important role in carbon
turnover and the ecology of this area. To characterize
the relationship between the fungus-growing termites,
their symbiotic fungi, and bacteria within the comb
and termite gut, we investigated the diversity of micro-
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C Institute of Zoology, Chinese Academy of Sciences, Insect Science, 17, 265–276
organisms under symbiotic conditions within colonies of
O. yunnanensis.
Materials and methods
Termites and fungi
The workers, fungus nodules, and fungus combs of
O. yunnanensis (Y3) were directly collected under asep-
tic conditions from a nest in Xishuang Banna, Yunnan
Province, China, in March 2008. All samples were quickly
frozen in liquid nitrogen in the field and then stored at
−80◦ C until use.
Isolation and cultivation of fungi
One fungus nodule was carefully grasped with sterile
forceps and gently washed three times with sterile distilled
water. The nodule was then aseptically inoculated onto an
improved potato dextrose agar plate (IPDA: 20% (w/v)
potato extract, 2% (w/v) glucose, 0.001% (w/v) VB1,
0.0003% (w/v) VB2, 0.0003% (w/v) VB6, 0.00002%
(w/v) biotin, 1.5% (w/v) agar, pH 4.5 regulated by cit-
ric acid) at 28◦ C. When white mycelium developed from
a nodule, it was further streaked and sub-cultured on a new
plate. This purification step was replicated at least three
times. The pure fungi were then inoculated into 100 mL of
improved potato dextrose liquid medium (IPD, the same
components with IPDA except no agar). The flasks were
incubated at 28◦ C for 5 days under constant shaking at
150 cycles per minute. The fragmented mycelial culture
(1%, v/v) was used as the inoculum for successive transfer
cultures.
DNA extraction
A FastDNA R
SPIN for Soil KIT (Qbiogene, Carlsbad,
CA, USA) was used to extract DNA of nodules and combs.
Five hundred (± 5) mg of nodules and combs of Y3 were
each prepared in 2-mL Lysing Matrix E tubes (supplied
by the Kit mentioned above) together with 978 μL of
sodium phosphate buffer and 122 μL of MT buffer. The
samples were treated with bead beating using a FastPrep R
Instrument (MP Biomedicals, Solon, OH, USA) for 30 s at
a speed setting of 5.5 m/s. The suspension was centrifuged
at 14 000 g for 30 s, and then the supernatant fluid was
continued according to the instructions provided with the
extraction kit.
The complete guts of five individual termites were
dissected and placed into phosphate buffer solution

C 2010 The Authors
 Comparison of micro-organism communities 267

(137 mmol/L NaCl, 2.7 mmol/L KCl, 10 mmol/L and electrophoresed on a 1% (w/v) agarose gel. The
Na2 HPO4 , 2 mmol/L KH2 PO4 ). For gut dissection, the purified products were sequenced by SUNNYBIO
head of the termite was removed using a sterile pin, and (Shanghai, China) on a 3730XL DNA Sequencer. Re-
then the entire gut was drawn out from the end of the ab- sulting sequences and other homologous sequences were
domen using sterile forceps. Total DNA was extracted us- aligned using Clustal X (Thompson et al., 1997) and
ing a QIAamp DNA Stool Mini Kit (QIAGEN, Hamburg, were adjusted by eye using MacClade 4.0 (Maddison &
Germany) according to the manufacturer’s instructions. Maddison, 2000). All sequences were used for the con-
The DNA of Y3 liquid-cultured mycelium was ex- struction of phylogenetic trees to determine the taxonomy
tracted and purified following Wang et al. (2005), except of the strain.
for the cell lysing procedure. The lyophilized mycelium
was entirely crushed in liquid nitrogen using a mortar
The construction of the ITS-rDNA and 16S-rDNA
instead of a spatula, and the powders were carefully trans-
libraries
ferred into a 1.5-mL Eppendorf tube with 1 mL of wash-
ing buffer (1% [w/v] polyvinyl pyrrolidone, 0.05 mol/L
Bacterial 16S rDNA genes were amplified using the
ascorbic acid, 0.1 mol/L Tris-HCl [pH 8.0], 2% (w/v)
universal primers 27F (5 -TAG AGT TTG ATC CTG GCT
β-mercaptoethanol). The suspension was centrifuged at
CAG-3 ) and 1392R (5 -GAC GGG CGG TGT GTA CA-
20 000 g for 3 min. The pellet was resuspended with
3 ) (Lane, 1991). The genomic DNA from host gut and
700 μL 2 × hexadecyl trimethyl ammonium bromide
fungus comb was also used as a template. PCR reactions
(CTAB) (100 mmol/L Tris-HCl, pH 9.0; 40 mmol/L ede-
began with an initial denaturation at 95◦ C for 3 min and
tic acid (EDTA), pH 8.0, 100 mmol/L sodium phosphate
were followed by five cycles consisting of denaturation
buffer, pH 8.0; 2% (w/v) sodium dodecyl sulfate (SDS);
(30 s at 94◦ C), annealing (30 s at 60◦ C), and extensi-
30% (w/v) benzylchloride), which differed from the ex-
on (4 min at 72◦ C); five cycles consisting of denaturation
traction buffer used by Wang et al. (2005). Subsequent
(30 s at 94◦ C), annealing (30 s at 55◦ C), and extension
steps were performed as described in Wang et al. (2005).
(4 min at 72◦ C); 10 cycles consisting of denaturation (30 s
The concentration and quality of the purified DNA were
at 94◦ C), annealing (30 s at 50◦ C), and extension (4 min at
evaluated by both 1% (w/v) agarose (Biowest, Madrid,
72◦ C), and a final extension at 72◦ C for 10 min. The PCR
Spain) gel electrophoresis and spectrophotometry.
product was visualized on a 1% (w/v) agarose gel stained
with ethidium bromide. ITS region genes of the rDNA
were amplified as described in the previous paragraph.
PCR amplification of the ITS region and identification of
The purified products were ligated into pMD18 T-
the strain
Vectors (TaKaRa, Dalian, China) by incubating at 16◦ C
overnight, in accordance with the manufacturer’s instruc-
Polymerase chain reaction (PCR) amplification of the
tions. The prepared TOP10 cells were transformed using
internal transcribed spacer (ITS) region of the riboso-
a heat shock protocol; plated onto Luria-Bertani (LB)
mal DNA (rDNA) gene fragment was performed using
agar containing ampicillin (50 μg/mL), isopropyl β-D-1-
a Flexigene thermal cycler (Flexigene TECHNE, Abing-
thiogalactopyranoside (IPTG) (20% w/v), and X-gal (2%
don, UK) with forward primer ITS1 (TCC GTA GGT
w/v) solution; and then incubated at 37◦ C overnight. The
GAA CCT GCG G) and reverse primer ITS4 (TCC TCC
selected white transformants were examined using PCR
GCT TAT TGA TAT GC) (Mackay et al., 2001). The
with the universal primers M13F (GTA AAA CGA CGG
different genomic DNA from host gut and fungus comb
CCA G) and M13R (CAG GAA ACA GCT ATG AC).
was used as template. Amplifications (50 μL) were per-
All positive transformants were selected for sequencing
formed using 10 ng of genomic DNA, 5 μL of 10 ×
and analysis.
PCR buffer (200 mmol/L Tris-HCl, pH 8.4; 2.5 mmol/L
MgCl2 ; 500 mmol/L KCl), 0.25 mmol/L each of deoxyri-
bonucleotide triphosphates (dNTPs), 0.2 mmol/L of each Phylogenetic analysis
primer, and 1 U of Taq DNA polymerase (TaKaRa, Dalian,
China). The following reaction program was used: initial Inspection of sequence chromatograms and assembly
denaturation cycle, 3 min at 94◦ C; 30 cycles of 45 s at of bi-directional sequences were conducted using a Se-
94◦ C, 45 s at 58◦ C, and 1 min at 72◦ C; and 1 cycle of quence Scanner v1.0 (Applied Biosystems, Foster City,
10 min at 72◦ C. Aliquots of each PCR product from these CA, USA) and CodonCode Aligner software (Codon
amplifications were purified using a QIAquick PCR Pu- Code, Dedham, MA, USA). The alignments were per-
rification Kit protocol (QIAGEN, Hamburg, Germany) formed with BioEdit (Hall, 1999) using the ClustalW


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268 Y. Long et al.
method. The aligned sequences were corrected manu-
ally, and DNA sequence data were analyzed to provide
pair-wise percentage sequence divergence. Diversity cov-
erage by the clone library was analyzed using Analyt-
ical Rarefaction software (version 1.4; S. M. Holland,
University of Georgia, Athens, GA, USA). Molecular
evolutionary analyses were conducted according to the
neighbor-joining method after 1 000 bootstrap replicates
using MEGA version 4 (Tamura et al., 2007). Using the
PAUP 4.0 b10 (Swofford, 2000) program package, the
maximum parsimony (MP) method was employed to con-
struct phylogenetic trees based on the sequences of the
16S rDNA gene. Bootstrap values for individual branches
were also estimated by bootstrapping with 1 000 repli-
cations. Rooted trees were generated using TREEVIEW
version 1.6.6 (Page, 1996).
Results
Identification of cultures
Using micromorphology alone, the Termitomyces nod-
ules were difficult to identify. Therefore, two sequences
for the ITS regions of a wild nodule and cultivated
mycelium (FJ769409, FJ769410) obtained through PCR
were aligned and molecular information was used for
identification. The length of ITS1-5.8S-ITS2 was moder-
ately large (∼670 bp), which was in accordance with the
value of 600–800 bp generally reported for fungi (Gardes
& Bruns, 1996). Consensus was observed between the
two sequences with almost entirely uniform sequences.
These results indicated that the wild nodule and cultivated
mycelium were the same species of fungus. We were also
able to concomitantly cultivate this strain of Termitomyces
in the laboratory.
To determine the taxonomy of an isolated strain from a
Y3 nest, a sequence of the ITS region from the wild nodule
(FJ769410) was aligned with other submitted sequences
downloaded from GenBank. To help distinguish the strain,
a phylogenetic tree was constructed using the sequencing
of ITS (Fig. 1). Comparison of the sequence similarity
showed that 21 strains were significantly related, and these
were grouped into two clusters. The Y3_nodule sample,
together with 15 other sequences, formed a larger clus-
ter with high support (100%). The clade composed of
Y3_nodule_ITS and Termitomyces sp. Group 8 was also
well supported (99%). Termitomyces sp. Group 8 was not
given an exact species name because of the imperfection
of the classification of the genus Termitomyces. Thus, the
strain isolated from the Y3 nest was simply identified as
Termitomyces sp.
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C Institute of Zoology, Chinese Academy of Sciences, Insect Science, 17, 265–276
Abundance of fungi in the comb and gut of Y3
The ITS-rDNA libraries of the fungus comb and gut
of Y3 were constructed separately. With the exception of
some inferior sequences, 87 and 79 available sequences
for the fungus comb and Y3 gut libraries, respectively,
were obtained for subsequent analysis. For the fungus
comb library, all sequences were aligned after the primer
segments were removed. The sequences generated were
identical to FJ769409/FJ769410 mentioned earlier. Us-
ing PCR of the ITS region, results indicated that only
Termitomyces sp. fungi were cultivated by termites on
the fungus comb. The pattern of the Y3 gut library
was nearly identical to that of the fungus comb. Inter-
estingly, two sequences similar to Aspergillus penicil-
lioides (AY373862) and Chlamydomonas allensworthii
(AF326852) were found; support for these identities was
95% and 82%, respectively. As far as we know, no previ-
ous studies have found these two types of fungi in the gut
of Odontotermes termites.
Abundance of bacteria in the comb and gut of Y3
To compare bacterial diversity between the termite di-
gestive tract and the fungus comb, two bacterial 16S
rDNA gene clone libraries were constructed with PCR
using the complete DNA extracted from the gut of the
termite and the fungus comb. After alignment and tests
for chimera, 83 and 66 available sequences (≈ 1.4 kb)
of the fungus comb and gut libraries, respectively, were
obtained from the web (http://greengenes.lbl.gov/cgi-
bin/nph-index.cgi). Suspect sequences were omitted from
subsequent analysis, resulting in 40 and 66 phylotypes
identified based on an arbitrarily defined criterion of
> 97% sequence identity in the fungus comb and gut li-
brary, respectively. Of the total 106 phylotypes in the anal-
ysis, 86 occurred only once. The representative sequences
of all phylotypes were submitted to the Ribosomal
Database Project (http://rdp.cme.msu.edu/index.jsp) for
classification and sequence matching (data not shown).
Figure 2 presents rarefaction curves for the phylotypes;
the number of samples for the gut library was not ade-
quate for analysis. However, the sample numbers for the
phylotypes of the fungus comb library were considered
saturated.
Figure 3 presents the relative clone frequencies of the
16S rDNA libraries of the fungus comb and Y3 gut.
The abundance of bacteria in the termite gut was much
higher than in the fungus comb. The basal bacterial com-
position of the two libraries was very similar, but the

C 2010 The Authors
 Comparison of micro-organism communities 269

Fig. 1 Phylogenetic tree of Termitomyces based on ITS1-5.8S-ITS2 complete sequence. Numbers above the branches indicate bootstrap
values of parsimony analysis from 1 000 replicates. The numbers that follow species names are the accession numbers for public
databases. The scale bar indicates 10% estimated sequence divergence.

proportions of major phyla were distinguishable. The
bacterial community structure of the fungus comb was
strongly dominated (77.5% of phylotypes) by bacterial
sequences affiliated with the Firmicutes phylum. How-
ever, clones of this phylum also represented the largest
component of the gut library, comprising approximately
38% of all assigned clones. Bacteroidetes (26.7%) were
identified as the second major constituent in the gut li-
brary, and this group only comprised 2.5% of the fungus
comb library. The sequences of Proteobacteria in the fun-
gus comb (15%) were slightly more abundant than in the
gut library (11.8%). Differences between the two libraries
were tested using P-test pair-wise comparisons (data not
shown). At the level of phylum, the two libraries exhib-
ited significant differences for Firmicutes, Bacteroidetes,
Proteobacteria, and Spirochaetes. Additionally, our anal-
ysis of the fungus comb library revealed the presence of
Actinobacteria clones (2.5%), which were not found in the
termite gut library. Moreover, no clones affiliated with the
phylum Spirochaetes were identified in the fungus comb


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C Institute of Zoology, Chinese Academy of Sciences, Insect Science, 17, 265–276
270 Y. Long et al.
Fig. 2 Rarefaction curves for phylotypes of 16S rDNA gene
clones of different samples. The expected number of phylotypes
was calculated from the number of clones, with inclusion in the
same species based on 97% sequence similarity.
library, whereas this group comprised 8% of phylotypes
in the gut library.
Because the fungus comb was dominated by the Firmi-
cutes phylum, we used the MP calculation method (incor-
porating 50 phylotypes of the two libraries) to construct
a dendrogram and to map bootstrap scores (Fig. 4). All
sequences of Firmicutes derived from the fungus comb or
gut formed four distinct groups. Groups I and III were
primarily composed of clones from the fungus comb,
Fig. 3 Relative clone frequencies of 16S rDNA libraries. A: fungus comb of Y3. B: gut of Y3.
Journal compilation 
C Institute of Zoology, Chinese Academy of Sciences, Insect Science, 17, 265–276
whereas groups II and IV were composed of clones from
the gut, with the exception of two clones. Clones de-
rived from the same origin clearly clustered together.
Groups I, II and III were considered Clostrididiales, but
Group IV remains unidentified. In Group I, the origin
of all clones was determined as Clostridium, except for
comb_2_08. This clone was clustered with L35515 (Ace-
tivibrio), which had only 61% support. Nearly half of the
Firmicutes clones from the fungus comb were derived
from Clostridium genera closely affiliated with two se-
quences from the termite gut in Group I or possibly with
all sequences of Group II.
Discussion
The entire ITS region of fungi often ranges between 600
and 800 bp and can be readily amplified using “univer-
sal primers” that are complementary to sequences within
rRNA genes (White et al., 1990). Several studies have
reported potential disadvantages when PCR-amplifying
DNA templates from a broad range of organisms, includ-
ing fungi, plants, protists and animals (White et al., 1990;
Gardes et al., 1991; Gardes & Bruns, 1991; Baura et al.,
1992; Chen et al., 1992; Lee & Taylor, 1992); despite this,
the method is still widely used in studies of fungal iden-
tification and phylogeny (Moriya et al., 2005; Shinzato

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 Comparison of micro-organism communities 271

Fig. 4 Molecular phylogenetic tree of 16S ribosomal RNA partial gene sequences associated with Firmicutes phylum using maximum-
pasimony analysis. Numbers above the branches indicate bootstrap values of parsimony analysis from 1 000 replicates. The numbers
that follow species names are the accession numbers for public databases.


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C Institute of Zoology, Chinese Academy of Sciences, Insect Science, 17, 265–276
272 Y. Long et al.
et al., 2005; Lefèvre et al., 2002; Aanen et al., 2007). In the
present study, the Y3_nodule_ITS sequence (FJ769410)
and other Termitomyces sp. sequences from fungus nod-
ules or fruit (downloaded from the National Center for
Biotechnology Information [NCBI], which were reported
in papers examining symbiotic fungi of termites) were sig-
nificantly related. Meanwhile, FJ769410 exhibited nearly
the same sequence as Termitomyces sp. Group 8 (Katoh
et al., 2002; Taprab et al., 2002), which had not yet been
given an exact species name. The two sequences also clus-
tered together with 99% support in the phylogenetic tree.
Additionally, due to the imperfection of classification in-
formation concerning the genus Termitomyces, we could
only identify this strain as Termitomyces sp. based on uni-
form experience of molecular identification using PCR
with available primers. Remarkably, the host termites of
these two strains are not the same. The host of Termito-
myces sp. Group 8 is M. annandalei (Katoh et al., 2002;
Taprab et al., 2002), whereas the cultures obtained in this
study were associated with another genus, Odontotermes.
A similar phenomenon was also demonstrated by Aanen
et al. (2007); because they found either similar or higher
diversity in the symbiont, the fungal symbiont clearly does
not always follow the general pattern of the endosymbiont.
One important result was that no other fungi except Ter-
mitomyces could be detected in the fungus comb using the
method of library construction of ITS. Similar patterns
have been observed by other researchers. For example,
using the method of terminal restriction fragment length
polymorphism (T-RFLP), Moriya et al. (2005) concluded
that the growth pattern of Termitomyces was a monocul-
ture, whereas non-Termitomyces inhabitants were only mi-
nor components in fungus gardens. Shinzato et al. (2005)
examined the growth state of fungi using laboratory-scale
fungus combs with or without termites under laboratory
conditions. They found that non-Termitomyces species
grew vigorously in combs without termites, although
no significant differences were observed in combs with
termites. The monoculture pattern of Termitomyces in
combs with termites was also confirmed using molecular-
based analyses of the microbial communities in the combs
(Shinzato et al., 2005). Recently, Guedegbe et al. (2009)
demonstrated that active combs were dominated by bands
of Termitomyces fungi, which were isolated using direct
polymerase endonuclease restriction–denaturing gradient
gel electrophoresis (PCR-DGGE). Taken together, these
previous studies confirm the credibility of our present
results.
However, in the studies mentioned above, non-
Termitomyces fungi, such as some filamentous fungi
and yeasts, were detected in laboratory culture (Shin-
zato et al., 2005) and by using the suicide polymerase
Journal compilation 
C Institute of Zoology, Chinese Academy of Sciences, Insect Science, 17, 265–276
endonuclease restriction (SuPER) PCR-DGGE method
(Guedegbe et al., 2009). During our experiment, we also
found that some filamentous fungi developed within the
combs (without termites) when they were removed from
the nests, whereas the growth of these non-Termitomyces
fungi was inhibited within the nests (with termites). Nev-
ertheless, no sequences of these filamentous fungi were
obtained from the ITS-rDNA gene clone library. The ab-
sence of such sequences may have occurred for several
reasons. The first reason is related to the methodology of
sample collection. In the field, the combs were quickly
removed from nests and placed into liquid nitrogen. Thus,
the samples used to construct libraries originated from the
most natural conditions possible, perhaps avoiding con-
tamination. Another possible reason could be due to the
relatively small screening size (87 clones). The lack of
filamentous fungal contamination may also be related to
why only the genus Termitomyces thrives on the fungus
comb with termites. For example, Macrotermitinae ex-
crete several fungicides (Bulmer & Crozier, 2004; Lam-
berty et al., 2001), although the range of sensitivity of
these fungicides has not yet been determined. Such fungi-
cides may have prevented contamination in the cultivation
of fungus gardens and helped to maintain the Termito-
myces monocultures (Moriya et al., 2005). Additionally,
antimicrobial substances derived from a Streptomyces
bacterium may have played a role in preserving uncon-
taminated conditions in colonies of the attine ant (Lam-
berty et al., 2001; Mueller & Gerardo, 2002; Shinzato
et al., 2005). Further studies are necessary to determine
whether special bacteria with similar functions are present
in the intestinal tract of termites. Finally, high concentra-
tions of CO2 in the nests may provide a competitive edge
for Termitomyces relative to non-Termitomyces species,
as Termitomyces may be more tolerant of high CO2
concentrations compared to other fungi (Batra & Batra,
1966).
In the comparison of bacterial microbiota in the termite
gut and fungus comb, the phyla of Firmicutes were sig-
nificantly dominant, followed by phyla of Bacteroidetes
and Proteobacteria in the gut and comb, respectively. The
diversity of bacterial flora of the gut was clearly higher
than that of the fungus comb (Fig. 3). Yet, the similar
pattern of floral structure could indicate strong proof that
the fungus comb was composed of termite feces. In the
gut, the abundant population of Clostridiales (Firmicutes)
was consistent with the results of studies of other fungus-
growing termites, such as Macrotermes gilvus (Hongoh
et al., 2006), Cubitermes niokoloensis (Fall et al., 2007),
and O. formosanus (Shinzato et al., 2007). Furthermore,
the cluster analysis of the Firmicutes clones from the two
libraries (Fig. 4) unequivocally indicated that clones from

C 2010 The Authors

the same sample clustered together even though they all
belonged to the same phylum. More than half of the Fir-
micutes clones from the fungus comb were Clostridiales
genera, which were closely affiliated with sequences from
the termite gut. Although most clones blasted as uncul-
tured bacteria that also originated from the termite gut or
environmental samples, the predominance of Clostridi-
ales, together with the Termitomyces fungi, likely aids
the termite host in the dissimilation of plant-derived ma-
terials (Anklin-Mühlemann et al., 1995; Hongoh et al.,
2006). The gut symbionts of various fungus-growing ter-
mite species, especially those of the genus Odontotermes,
are often comprised of similar bacterial communities. This
phenomenon can be explained by the theory that gut mi-
crobes, which co-evolved with termites, were transferred
via vertical transmission (Shinzato et al., 2007). Addi-
tionally, spirochetes are often a major constituent of the
bacterial flora of the guts of wood-feeding termites but
not of fungus-growing termites (Leadbetter et al., 1999;
Lilburn et al., 1999). Similarly, the population of spiro-
chetes (8%) observed in the gut of our termites also com-
prised a small fraction compared to that observed in wood-
feeding termites. A similar result has also been reported
for O. formosanus (Shinzato et al., 2007). The existence
of such a relatively minor component of this phylum in
fungus-growing termite guts was likely of minimal phys-
iological significance. In the analysis of the two libraries,
2.5% of phylotypes of the fungus comb were identified
as a type of acidophilic bacteria (Acidobacteria), which
play an important role in ecological systems, yet remain
understudied. The presence of these bacteria suggests that
the pH of the fungus comb should be further examined
to learn the exact association between the comb and the
gut. However, no clones of this phylum were found in the
termite gut, perhaps due to the fact that fewer phylotypes
were obtained during the construction of the 16S rDNA
library of the termite gut.
The observed bacterial community of the fungus comb
distinctly differed from those reported previously. For ex-
ample, Fall et al. (2007) found that Actinobacteria phy-
lotypes dominated in the termite mound. In contrast, we
found that Firmicutes were dominant in the combs. This
discrepancy may have resulted from the use of different
genera of termites (Cubitermes and Odontotermes), the
limited size of screened clones, or differences in sam-
ple origin (ours was the fungus comb composed of fe-
ces and lignin materials, whereas the other was an in-
ternal mound wall consisting of soil). In fact, Fall et al.
(2007) did observe a shift in the bacterial community
structure between the termite gut and mound; this change
was obvious even in samples of fresh feces sampled
< 1 h after being deposited in the mound. The nature

C 2010 The Authors
Journal compilation 
C Institute of Zoology, Chinese Academy of Sciences, Insect Science, 17, 265–276
Comparison of micro-organism communities 273
of such rapid bacterial community shifts deserves further
investigation.
No gene clones associated with the degradation of
lignocelluloses were found in either the gut or fungus
comb library because the current research did not focus
on lignocellulase identification, whereas genes obtained
through the 16S-rDNA library were strongly related to
metabolism. Within such a complex symbiotic system
composed of a termite, bacterium and fungus, the fun-
gus could possibly play a crucial role in the degrada-
tion of lignocelluloses. There is evidence in the literature
of lignocellulases in Termitomyces fungi. For example,
several genes or enzymes have been cloned or purified
from the Termitomyces fungus, such as laccase (Taprab
et al., 2005; Bose et al., 2007), phenol oxidase (Johjima
et al., 2003a), cellobiase (Mukherjee & Khowala, 2002;
Mukherjee et al., 2006), amyloglucosidase (Ghosh et al.,
1997), and xylanase (Matoub & Rouland, 1995). The cur-
rent study provides a step in the process of identifying
novel fungal lignocellulases. Thus, future studies should
seek to identify lignocellulase-coding genes from fungal
symbionts.
Acknowledgments
This manuscript benefited greatly from comments by
Prof. Xiangxiong Zhu, Ms. Yixin Zhang, and two anony-
mous referees. This study was supported by the national
High Tech 863 Project (2007AA021302), a grant from the
Knowledge Innovation Program of the Chinese Academy
of Sciences (KSCX2-YW-G-022, KSCX2-YW-G-062),
the Knowledge Innovation Program of the Shanghai In-
stitutes for Biological Sciences, Chinese Academy of Sci-
ences (2007KIP501), and also in part by the Anhui Col-
leges Young Teachers’ Funded Projects (2008jq1051).
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Accepted February 1, 2010

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 Insect Science (2010) 17, 277–290, DOI 10.1111/j.1744-7917.2010.01336.x

ORIGINAL ARTICLE

In-situ oxygen profiling and lignin modification in guts

of wood-feeding termites

Jing Ke1 , Jian-Zhong Sun2,3 , Hung D. Nguyen4 , Deepak Singh1 , Karmen C. Lee2 , Haluk Beyenal4
and Shu-Lin Chen1
1 2
Department of Biological Systems Engineering, Washington State University, Pullman, Washington, Coastal Research and Extension
Center, Mississippi State University, Poplarville, Mississippi, USA, 3 School of the Environment, Jiangsu University, Zhenjiang, Jiangsu
Province, China, 4 The Gene and Linda Voiland School of Chemical Engineering and Bioengineering, Washington State University,
Pullman, Washington, USA

Abstract Reports on the capability of wood-feeding termites (WFTs) in degrading wood

particles and on the existence of aerobic environment in the localized guts suggest that
their high efficiency of cellulose utilization is not only caused by cellulase, but also by
biochemical factors that pretreat lignin. We thus extend the hypothesis that for highly
efficient accessibility of cellulose, there should be direct evidence of lignin modification
before the hindgut. The lignin degradation/modification is facilitated by the oxygenated
environment in intestinal microhabitats. To test our hypothesis, we conducted experiments
using a dissolved oxygen microelectrode with a tip diameter < 10 μm to measure oxygen
profiles in intestinal microhabitats of both Coptotermes formosanus (Shiraki) and Reti-
culitermes flavipes (Kollar). Lignin modification during passage through their three gut
segments was also analyzed with pyrolysis gas chromatography/mass spectrometry. The
data showed relatively high levels of oxygen in the midgut that could have promoted lignin
oxidation. Consistent with the oxygen measurements, lignin modifications were also de-
tected. In support of previously proposed hypotheses, these results demonstrate that lignin
disruption, which pretreats wood for cellulose utilization, is initiated in the foregut, and
continues in the midgut in both termites.
Key words Coptotermes formosanus, lignin modification, oxygen profile, pretreatment,
Reticulitermes flavipes, wood-feeding termites (WFTs)

Introduction strength to plant cell walls and protect other structures

Lignin, the second most abundant organic polymer in na-
ture, is covalently linked to hemicellulose and crosslinks
different plant polysaccharides to confer mechanical
Correspondence: Shu-Lin Chen, Department of Biological
Systems Engineering, PO Box 646120, Washington State Uni-
versity, Pullman, WA 99164-6120, USA. Tel: +1 509 335 3743;
fax: +1 509 335 2722; email: chens@wsu.edu
Jian-Zhong Sun, School of the Environment, Jiangsu Uni-
versity, Zhenjiang, 212013, China. Tel: +86 0511 88796122;
e-mail: jzsun1002@hotmail.com
from being damaged by external factors (Boerjan et al.,
2003; Chabannes et al., 2001). On the other hand, lignin
is heterogeneously and irregularly constructed of cross-
linked phenylpropanoid monomers of cinnamyl alcohol
derivatives (Goodell et al., 2003). The phenylpropanoid
polymer resists chemical, enzymatic and microbial degra-
dation (Tuomela et al., 2000), and is covalently linked
at various points with hemicellulose to form a matrix
surrounding the orderly cellulose microfibrils (Jeffries,
1990). This arrangement results in resistance to saccha-
rification, thus severely limiting biochemical conversion
of lignocellulosic biomass to ethanol (Chen & Dixon,
2007) and other biofuels. Realization of commercial


C 2010 The Authors 277
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C Institute of Zoology, Chinese Academy of Sciences
278 J. Ke et al.
success for lignocellulosic ethanol production will require
advanced pretreatment methods that efficiently remove
protective lignin from cellulose (Mosier et al., 2005).
Current chemical and physical pretreatment methods suf-
fer from high costs and undesirable environmental im-
pacts. Alternative biological pretreatments, such as that
by white- and brown-rot fungi are often too slow to
be economically feasible (Filley et al., 2000; Martinez
et al., 2008). However, lignocellulose-feeding insects, es-
pecially wood-feeding termites (WFTs), have proven to
be a promising model organism for biological pretreat-
ment of biomass due to their highly efficient digestion of
wood (65%–99% wood-cellulose and hemicellulose uti-
lization within 24 h) under natural conditions (Esenther
& Kirk, 1974; Wood, 1978; Brune, 2007). The plant cell
wall degradation processes in such systems may provide
another potential alternative for biomass processing.
For a long time, little has been revealed about lignin
modification in termites, but it has been thought that host-
derived enzymes and/or a consortium of micro-organisms
may be involved in degradation (Scharf & Tartar, 2008).
Recently, WFTs’ high efficiency in wood digestion has at-
tracted researchers’ attentions (Sun & Zhou, 2010); how-
ever, most research has focused on the cellulase system.
To fully understand wood degradation in termites, the
pretreatment system must also be addressed. Kyou et al.
(1996) pointed out that wood lignin plays a role in main-
tenance of health in workers of Coptotermes formosanus
Shiraki. French and Bland (1975) inferred lignolysis for
Coptotermies lacteus and Nasutitermies exitiosus by mea-
suring incorporation of 14 C-label into tissues of termite
fed wood previously grown with [3-14 C] cinnamic acid.
Butler and Buckerfield (1979) reported that lignin degra-
dation occurs within termites’ bodies, and not in voided
fecal pellets, although the site of degradation was not
identified, and the relevance of gut organisms in the
process was not addressed (Breznak, 1982). Katsumata
et al. (2007) suggested there were some changes in the
structural features of lignin during digestion by termites.
Later, Geib et al. (2008) proved that there were significant
levels of propyl side-chain oxidation (depolymerization),
demethylation of ring methoxyl group, and ring hydroxy-
lation on lignin after passage through the gut of a Pacific
dampwood termite (Zootermopsis angusticollis). The au-
thor demonstrated the lignin degradation/modification
process occurs in the whole gut environment. Scharf and
Tartar (2008) summarized that significant lignin degra-
dation, which should be the first step in the process of
lignocellulose degradation, can take place in the termite
gut; and Tartar et al. (2009) recently confirmed this state-
ment by identifying lignase gene candidates in the Reti-
Journal compilation 
C Institute of Zoology, Chinese Academy of Sciences, Insect Science, 17, 277–290
culitermes gut. In all likelihood, the success of WFTs
in wood-cellulose digestion can not only be attributed
to cellulase, but also to pretreatment factors that mod-
ify lignin and increase accessibility of wood-cellulose.
Lignin degradation by WFTs is intriguing but may be
ambiguous in some way, because the termite gut used
to be considered as anaerobic on the basis of presence
of strictly anaerobic micro-organisms in termite hindgut
(Eutick et al., 1976; Breznak, 1982; Katsumata et al.,
2007), but high oxygen tension is required for lignin
degradation (Ten Have & Teunissen, 2001; Scharf &
Tartar, 2008). In an anaerobic environment, polymeric
lignin will not be degraded, and wood may persist in
non-degraded form for several hundreds or thousands
years (Blanchette, 1995). The presence of molecular oxy-
gen is a prerequisite for the initiation of lignin depoly-
merization since oxygen acts as a co-substrate in the
modification/disruption of lignin by oxidative enzymes
(Breznak & Brune, 1994; Scharf & Tartar, 2008). There
is no conclusive evidence that the inter-monomeric link-
ages in insoluble, highly polymeric lignins are attacked in
the absence of oxygen (Colberg, 1988; Young & Frazer,
1987).
For this study, we hypothesized that WFTs have evolved
exactly the right distribution of oxygen in gut microhab-
itats to facilitate a process of enzymatic modification on
less recalcitrant lignin linkage, and thus increase accessi-
bility of cellulose. To test the hypothesis, we studied two
species of lower subterranean WFTs within the same fam-
ily of Rhinotermitidae, Coptotermes formosanus (Shiraki)
and Reticulitermes flavipes (Kollar). These two WFTs
have been the subjects of much research on termite control
due to their highly efficient digestion capability of cellu-
lose, assisted by the cellulolytic protozoa in the hindgut
(Tokuda et al., 1999; Shelton & Grace, 2003). Of these
two well-known termite species, C. formosanus, just pos-
sesses three species of cellulolytic protozoa for cellulose
digestion (Yoshimura et al., 1993; Hu & Zhu, 2003); while
the other one, eastern subterranean termite, R. flavipes,
holds more than 10 types of cellulolytic protozoa to as-
sist wood degradation. These two termite genera have
world-wide distribution and are important structural pests.
Thus, we exploited the advances in microelectrode tech-
nology (Revsbech & Jørgensen, 1986; Revsbech, 1989;
Gross et al., 2008) to obtain oxygen profiles of the guts
from these two well-known termite species, and investi-
gated where the lignin oxidation might occur. We then
correlated these oxygen profiles with actual lignin modi-
fication during passage through WFT gut segments mea-
sured with pyrolysis gas chromatography/mass spectrom-
etry (Py-GC/MS).

C 2010 The Authors
 Oxygen profiling and lignin modification in termite 279

Materials and methods

Sample collection and preparation

Two species of WFTs, C. formosanus and R. flavipes,

collected in Poplarville, Mississippi, were fed at 28◦ C,
90% humidity on 6.8 × 1.5 × 0.5 inches of Southern pine
(Pinus australis F. Michx) blocks, the lignin of which
consists almost exclusively of guaiacyl propane subunits.
Isolation of termite guts was performed under a dis-
section microscope. The body wall of each termite was
opened and pulled aside to expose the intact gut. Then,
the gut was extended to allow identification of different
segments.

In-situ measurements of oxygen concentration profiles in

termite gut

Construction of microelectrode A dissolved oxygen

microelectrode (DOM) is an amperometric device in
which oxygen diffuses through a silicone rubber mem-
brane, arrives at a cathodically polarized working elec-
trode, and is reduced to water (Fig. 1). The device uses
an Ag/AgCl half-cell as the counter-electrode and a no-
ble metal of gold as the working electrode. Applying
−0.8 V typically satisfies the limiting current condition,
and the measured current is proportional to the dissolved
oxygen concentration in the vicinity of the sensor tip
(Lewandowski & Beyenal, 2007). Construction of the dis-
solved oxygen microelectrode consisted of the following
steps: (i) making an outer case; (ii) opening the tip of the
outer case; (iii) applying a silicone rubber membrane; (iv)
etching the Pt wire to a tip with a diameter of a few mi-
crometers; (v) making a shaft using Corning 8161 glass;
(vi) sealing the Pt wire in the shaft; (vii) recessing the tip
of the shaft; (viii) electroplating the tip of the Pt wire with
gold; (ix) making the electrical connections; (x) prepar-
ing the Ag/AgCl reference electrode; (xi) assembling all
of the components; (xii) filling the outer case with the
electrolyte; and (xiii) calibrating the microelectrode. The
entire process of the construction is given in detailed in
Lewandowski & Beyenal (2007).
Measurement of oxygen profiles Each oxygen profile
measurement was replicated three times using three dif-
ferent worker termite guts for each termite species. Each
termite gut sample was used for just one measurement to
prevent the diffusion of oxygen caused by gut wall dam-
age by the tip of the microsensor. After each dissection,
the isolated termite guts were placed on a dissection plate
and quickly coated with a thin layer of 1% agarose in
Fig. 1 Dissolved oxygen microelectrode (DOM). Reproduced
from Lewandowski and Beyenal (2007).
Ringer’s solution to fix the gut and restrict the diffusion
of oxygen for the conservation of the oxygen state in the
gut. The small amount of 65◦ C low melting point agarose
was used in an effort to avoid the gut wall and hindgut
symbiotic microbiota damage. During the measurements,
the isolated gut incubated in the argrose retained normal
muscular activity (peristalsis) for at least half an hour
(Brune et al., 1995a). All readings were completed within
10 min of dissection. We isolated the intact gut, thus ac-
quiring the opportunity to study the foregut, which is a
location of host phenol-oxidizing laccases (Tartar et al.,
2009).
The DOM had a tip diameter < 10 μm, response times
of 1–3 s, and stirring sensitivities of 0–1%. Prior to use,
the microelectrodes were calibrated using two-point cal-
ibration: in the air (oxygen saturation) and in saturated
Na2 SO3 solution (zero oxygen concentration). The work-
ing electrode was polarized by applying −0.8 V against
Ag/AgCl electrode using a picoammeter (HP 4140B). To
determine the spatial distribution of oxygen, one point of
penetration was measured in the foregut, and three points
each in the midgut and hindgut (paunch and colon). The


C 2010 The Authors
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C Institute of Zoology, Chinese Academy of Sciences, Insect Science, 17, 277–290
280 J. Ke et al.
scan of oxygen concentrations during each penetration
began from a little above the surface of the intestinal wall,
continued through the gut, and ended with emergence of
the DOM on the opposite side of the agarose layer. Mi-
croelectrode movements were controlled by a mercury-
step stepper motor (PI M-230.10S) with 10 ± 0.04 μm

R
step size using custom-made software Microprofiler
(The Gene and Voilland School of Chemical Engineer-
ing, Washington State University, Pullman, WA, USA).
Data was recorded in the computer using a data logger
(Measurement COMPUTING(tm) USB-1608FS: Mea-
surement Computing Corporation, Norton, MA, USA).
All measurements were carried out at ambient tempera-
ture (28◦ C).
Pyrolysis and GC/MS analysis of lignin modification
during passage through different gut segments of termites
Modifications of lignin during passage through each
gut segment were determined from lignin chemical struc-
ture. Pyrolysis gas chromatography/mass spectrometry
(Py-GC/MS) was used for this purpose. Py-GC/MS is
a rapid and highly sensitive method for characterizing
the chemical structure of lignin, which allows the anal-
ysis of very small amounts of sample without prior ma-
nipulation and isolation (Rı́o et al., 2004). Pyrolysis of
pinewood lignin yields a range of products, of which the
most characteristic are those solely based on guaiacol and
its derivatives. Although there is plenty of information
on the analytical pyrolysis of different wood types (Yokoi
et al., 2001; Schwarzinger et al., 2008), reports on bio-
logically modified wood are scarce.
The wood particle samples used in this study were ob-
tained from different gut segments: foregut, midgut and
hindgut, separated by scalpel; each piece of feces was col-
lected at the anus of the termite before each dissection.
Three foreguts, three midguts, two hindguts, and three
pieces of feces were used for each sample, respectively.
Sizes of the three gut segments are scaled. The guts of ter-
mites that fed on filter paper (C. formosanus) or mixture
of α-cellulose and avicel (R. flavipes) for 1 month were
employed as lignin-free guts. The powder of undigested
pinewood was employed as control. Samples were quickly
frozen in liquid N2 to halt lignin digestion, and then put
directly into a quartz tube (sample tube). The pyrolysis
processes were performed with a CDS 5000 pyrolysis
autosampler (CDS Analytical, Inc., Oxford, PA, USA)
attached to a Thermo Trace GC 6890N/MSD 5975B gas
chromatography/mass spectrometry system Agilent Tech-
nologies, Inc., Bellevue, WA, USA). The samples were
first pretreated at 210◦ C for 3 min, and then pyrolyzed at
Journal compilation 
C Institute of Zoology, Chinese Academy of Sciences, Insect Science, 17, 277–290
a temperature of 500◦ C for 1 min, and finally kept in the
pyrolysis zone for 56 min. The volatile products were sep-
arated on a 30 m × 0.25 μm inner diameter (5% phenyl)-
methylpolysiloxane non-polar column with helium 4.6 as
carrier gas (17.3 mL/min) and identified by interpretation
of their electron-impact (EI) mass spectra and comparison
to National Institute of Standards and Technology (NIST)
Mass Spectral (MS) Search 2.0 electronic libraries. The
pyrolysis interface was kept at 210◦ C, the GC/MS inter-
face at 280◦ C; the GC/MS was programmed from 40◦ C
(1 min) to 280◦ C (15 min) at a rate of 6◦ C/min. The mass
spectrometer was operated in EI mode (70 eV) at a source
temperature of 230◦ C.
Results
Distribution of oxygen in termite gut
Oxygen concentrations in each region of the termite
guts measured demonstrates that the morpho-anatomical
differentiation of the intestinal tract corresponds to differ-
ent oxygen concentrations, which will most likely create
different physicochemical microenvironments.
Radial distribution of oxygen in different segments
of termite guts Figures 2 and 3 show radial oxygen con-
centration profiles with three times replication in three
different worker termite guts for each species in two wood-
feeding termites (WFTs) C. formosanus and R. flavipes.
However, the data are just based on a single replicate
since the measurement started from an arbitrary selected
location near the gut surface and the distance of the micro-
electrode tip could not be quantize unified. The oxygen
concentration profiles were asymmetric for both cases
showing penetration of the tip of the microelectrode to
agar/gut/agar, which might be because of the different
thicknesses and compositions of the agar above and be-
low to lead to different degrees of oxygen penetration to
the gut. All the measured profiles clearly demonstrated
that the microhabitats were aerobic, which is in accor-
dance with the earlier result of Brune et al. (1995a); but
because we placed a thin layer of agar above the gut to
mimic termite body instead of their microchamber for nor-
mal oxygen penetration, this likely resulted in the average
oxygen concentration being higher than the result previ-
ously published (Brune et al., 1995a). Compared to Brune
et al. (1995a), our microsensor was apparently more ad-
vanced in tip sensitivity to be able to give precise oxygen
distribution along the whole gut, including foregut, to bet-
ter reveal the lignin oxidation situation in each segment.
The gut segments also showed different oxygen gradients

C 2010 The Authors

Fig. 2 Radial oxygen profiles along the gut of C. formosanus.
The relative distance refers the position relative to initial start-
ing location. Initially the measurements were started from an
arbitrary selected location near the surface. The data are based
on a single replicate since the distance of the microelectrode

C 2010 The Authors
Journal compilation 
C Institute of Zoology, Chinese Academy of Sciences, Insect Science, 17, 277–290
Oxygen profiling and lignin modification in termite 281
indicating different oxygen penetration or consumption
rates in these gut segments. Figure 2A shows a slow de-
cline of the radial oxygen concentration in the foregut
of C. formosanus; similar decreasing gradients were ob-
tained for R. flavipes (Fig. 3A). In both foreguts, there
were zones of abrupt oxygen transition (marked by solid
arrowheads).
The midguts of both WFTs contained significant
amounts of oxygen, despite their small diameters
(Figs. 2B and 3B), and they exhibited a more gradual
loss of oxygen than the hindguts did. For C. formosanus,
the anterior and posterior portions of the midgut revealed
steeper oxygen gradients than the middle region; while
the difference in R. flavipes seemed not so obvious.
In the three hindgut regions of both C. formosanus
and R. flavipes (Figs. 2C and 3C), there were linear de-
creases and then increases in oxygen concentrations from
the ambient concentrations at the agarose surface toward
the gut. Only the central region of the paunch was rela-
tively anoxic, with oxygen concentration of approximately
1 mg/L. This was most obvious in the second region of the
paunch. The oxygen gradient in the hindgut of R. flavipes
was less sharp, and exhibited more oxygen than that of C.
formosanus. The slopes of the oxygen gradient were quite
reproducible for all regions.
Axial distribution of oxygen in the gut of termites
from foregut to hindgut The anterior-to-posterior oxy-
gen profiles of termite guts were determined by measur-
ing the concentration in the center of each gut region with
three times replication in three different worker termite
guts for each species (Fig. 4). The error bar and averages
were derived from single measurements from multiple ter-
mites. Because of the artificial restriction of oxygen flux
toward the gut by the surrounding agarose, the measured
oxygen concentrations might be slightly different from
that in vivo (Ebert & Brune, 1997; Brune et al., 1995a).
Thus, the axial oxygen profiles shown in Fig. 4 represent
conservative estimates of the oxygen profiles in the guts
←−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−
tip could not be quantize unified. The hollow arrowheads indi-
cate the points at which the tip entered the gut wall or emerged
from the opposite side; the solid arrowheads indicate the points
of inflection of oxygen profiles. (A) Profiles obtained from a
typical foregut. (B) Profiles obtained in the midgut, illustrating
the difference among the median midgut (II), posterior (I), and
anterior (III) regions. (C) Profiles for the hindgut, illustrating
the differences among the colon (III), posterior paunch (I) and
anterior paunch (II) regions. Note the much sharper decrease of
oxygen profiles than those in foregut and midgut.
282 J. Ke et al.
Fig. 3 Radial oxygen profiles along the gut of R. flavipes.
The relative distance refers to the position relative to initial
starting location. Initially the measurements were started from
an arbitrary selected location near the surface. The data are based
on a single replicate since the distance of the microelectrode tip
Journal compilation 
C Institute of Zoology, Chinese Academy of Sciences, Insect Science, 17, 277–290
of C. formosanus and R. flavipes. However, they do illus-
trate the relative oxygen status in various gut regions. For
both termites, even the center of the hindgut, which had
the least oxygen, was not definitely anoxic. C. formosanus
exhibits a more rapid decrease in oxygen along the axis
of the gut than R. flavipes.
Lignin modification in different gut segments
Differences in gut segment sizes and food intake af-
fected the amount of wood particles in gut segments of
the two termite species tested. To estimate lignin modi-
fication of each sample, we calculated the proportions of
pyrolysed lignin compounds in the total aromatic com-
pounds. Twenty-three percent of all aromatic compounds
were contained in the pyrolysis products from the undi-
gested wood sample: 6%, 12%, 12%, and 41% in the C.
formosanus sample of foregut, midgut, hindgut and feces,
respectively; and 20%, 6%, 31%, 37% for R. flavipes,
respectively.
Figures 5 and 6 show pyrograms obtained from gut
samples of C. formosanus and R. flavipes, and the undi-
gested wood control, after being pyrolyzed at 500◦ C for
1 h. Meanwhile, cellulose-fed termite guts proved to be
lignin-free (Figs. 7 and 8). Lignin-derived compounds
were concentrated after passage through the termite gut in
comparison to undigested samples, mainly because of the
degradation of cellulose and hemicellulose. The aromatic
compounds concentrated in the feces of both termites were
similar, indicating similar mechanisms were in process in
the two species. These compounds were guaiacol, vinyl
guaiacol, isoengenol, 4-acetyl-2-methoxyphenol, vanil-
lyl methyl ketone and coniferyl alcohol. The two com-
pounds with aldehyde groups, 4-acetyl-2-methoxyphenol
and vanillyl methyl ketone, were not observed until
the hindgut, and seemed to reflect byproducts from the
lignin degradation process. This was consistent with the
suggestion that oxidation occurs at hydroxyl groups on
←−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−
could not be quantize unified. The hollow arrowheads indicate
the points at which the tip entered the gut wall or emerged
from the opposite side; the solid arrowheads indicate the points
of inflection of oxygen profiles. (A) Profiles obtained from a
typical foregut. (B) Profiles obtained in the midgut, illustrating
the differences among the median midgut (II), posterior (I) and
anterior (III) regions. (C) Profiles for the hindgut, illustrating
the differences among the colon (III), posterior paunch (I) and
anterior paunch (II) regions. Note the much sharper decrease of
oxygen profiles than those in foregut and midgut.

C2010 The Authors
 Oxygen profiling and lignin modification in termite 283

Fig. 4 Axial oxygen distribution in guts. The guts of both termites, C. formosanus (A) and R. flavipes (B), included the foregut (F),
midgut (M) and hindgut (H). The hindgut consisted of paunch (Pa), colon (Co) and rectum (Re). Since the rectum is not thought to be
an important digestive part for lignin modification, it has not been included. The reported oxygen concentrations were determined at
the center of each gut region with three times replication in three different worker termite guts for each species.

lignin polymer side-chains to generate aldehyde groups isoeugenol (prophenyl group) levels were similar to that
(Gvozdev & Chupka, 2004). Of the other four aro- of the control; coniferyl alcohol decreased in R. flavipes.
matic compounds pyrolyzed from the feces, only guaia- The increase in the methoxylated aromatic compounds
col obviously increased; vinyl guaiacol (vinyl group) and (guaiacol) in the feces may indicate loosely packed lignin

Fig. 6 Lignin modification during passage through gut of R.

Fig. 5 Lignin modification during passage through gut of C.
formosanus. (A) Control sample; (B) samples from foregut; (C)
samples from midgut; (D) samples from hindgut; (E) feces.
Chromatograms show increases in phenol (8.873 min), and p-
cresol (11.223 min) from the foregut (B), and further increase
in the midgut (C); as well as decreases in methyl guaiacol
(14.100 min), coniferyl alcohol (25.453 min). The various py-
rolysis products from hindgut sample (D) were mainly guaiacol;
but only several were left in the feces (E).
flavipes. (A) Control sample; (B) samples from foregut; (C)
samples from midgut; (D) samples from hindgut; (E) feces.
Chromatograms show obvious increases in phenol (8.873 min),
and p-cresol (11.223 min), and decreases in methyl guaiacol
(14.100 min), and coniferyl alcohol (25.453 min), in termite-
treated wood after passage through foregut (B) and midgut (C).
The pyrolysis products of the hindgut sample (D) were mainly
guaiacol (11.563 min), vinyl guaiacol (16.923 min) and 5-allyl-
2-methoxyphenol (17.897 min). Acetoguaiacon (20.651 min)
and vanillyl methyl ketone (21.554 min) increased in the feces
(E).


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284 J. Ke et al.

Fig. 7 Filter paper modification during passage through gut of Fig. 8 Filter paper modification during passage through gut
C. formosanus. (A) Filter paper; (B) samples from foregut; (C) of R. flavipes. (A) Filter paper; (B) samples from foregut; (C)
samples from midgut; (D) samples from hindgut. Few phenol- samples from midgut; (D) samples from hindgut. Few phenol-
related compounds showed in each sample. related compound showed in each sample.

These findings indicate lignin ring demethylation, decar-

morphology after lignin modification in termite guts
boxylation and side-chain oxidation (depolymerization)
which could facilitate the gasification process during py-
in the foregut and midgut.
rolysis, which may indicate carbon-carbon bond or al-
When passing through the hindgut, phenol, p-
lyl alcohol group cleavage, or some other conversion
cresol, 1,2-diformyloxyethane, and dimethyl 3,8-
of the hydroxyl group. The other guaiacol compounds
dioxodecanedioate maintained their concentrations
(methyl guaiacol, eugenol, 5-allyl-2-methoxyphenol, ho-
(Fig. 5D, Fig. 6D), but vanished in the feces. This was
movanillic acid, 4-hydroxy-2-methoxycinnamaldehyde)
consistent with the consumption of simpler products from
disappeared in the pyrolysis product from the feces, which
lignin degradation in the hindgut by anaerobic aromatic
suggested that methyl and allyl groups on aromatic rings,
lignin digesters, as suggested by Breznak and Brune
as well as carboxyl, aldehyde, hydroxyl groups on side-
(1994). Other guaiacol derivatives, such as methyl gua-
chains, had been modified or removed. The increase of
iacol, vinyl guaiacol, isoeugenol and coniferyl alcohol,
guaiacol, decrease of guaiacol derivatives, and appearance
initially decreased in midgut samples, but increased in
of new guaiacol derivatives after termite pretreatment sug-
hindgut samples and maintained their relative contents in
gested that there should be conversions among guaiacol
feces. Thus, conversions of functional groups on aromatic
and its derivatives in the gut, presumably as a result of
rings are likely to occur in the hindgut.
wood-lignin degradation. However, the fact that the con-
The hindgut pyrograms of the two WFTs showed dif-
centrations of aromatic compounds in the two WFTs fed
ferent levels of furfurol (retention time: 5.3–6.0 min) and
on the same diet were not identical indicates that the quan-
levoglucosan (20.5–21.0 min), which likely came from
tity and efficiency of enzymes or gut symbionts effecting
hemicellulose and cellulose, respectively. These results
lignin modification differ in the two species.
may be indicative of different digestion levels of hemi-
Changes in lignin content with progression through the
cellulose and cellulose in hindguts of the two species;
termite gut were observed, and provide some insight into
and R. flavipes appeared to possess higher efficiency in
the modification processes active in each gut segment.
holocellulose digestion.
Gut samples from C. formosanus (Fig. 5, Table 1) and
R. flavipes (Fig. 6, Table 1) exhibited similar trends for
all compounds. Compared to the control sample, 1,2- Discussion
diformyloxyethane, dimethyl 3,8-dioxodecanedioate and
p-cresol were increased in the foregut, and further in- This study has provided information on concentrations
creased when passing through the midgut. Phenol was of oxygen and lignin-derived compounds in various seg-
observed in the midgut samples. Guaiacol was found ments of the guts from two wood-feeing termite species.
to be increased in the midgut, whereas there were de- Compared to Brune et al. (1995a) and Ebert and Brune
creases of guaiacol derivatives, such as methyl guaiacol, (1997)’s work on oxygen profiles in R. flavipes gut, we
5-allyl-2-methoxyphenol, homovanillic acid, 4-hydroxy- advanced the microelectrode with smaller diameter and
2-methoxycinnamaldehyde, and coniferyl alcohol there. increment to be more accurate to reflect oxygen profiles


C 2010 The Authors
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C Institute of Zoology, Chinese Academy of Sciences, Insect Science, 17, 277–290
 Oxygen profiling and lignin modification in termite 285

Table 1 Percentage of pyrolysis products from lignin before and after biological modification by C. formosanus and R. flavipes

Retention undigested Foregut Midgut Hindgut Feces

Compounds
time (min) wood (%) (%) (%) (%) (%)

1,2-diformyloxyethane 4.120 1.228 1.414 1.994 1.857 ׆

1.611 1.431 1.597 ×
Phenol 8.873 0.089 × 1.228 1.137 ×
3.115 1.115 1.441 0.140
p-cresol 11.223 × 1.561 2.006 1.524 ×
5.326 1.663 2.141 0.126
Guaiacol 11.563 1.739 1.381 3.236 1.851 5.914
2.778 0.349 6.146 10.295
Dimethyl 3,8-dioxodecanedioate 13.482 0.109 0.218 0.596 0.444 ×
× 0.285 1.356 ×
4-methyl guaiacol 14.100 2.297 × 0.338 0.905 2.457
0.922 × 1.910 2.133
Eugenol 16.130 0.560 0.335 0.329 0.433 1.071
× 0.285 0.686 1.183
4-vinyl guaiacol 16.923 2.893 0.447 1.772 1.671 7.951
2.562 × 5.366 7.751
5-allyl-2-methoxyphenol 17.897 0.870 × 0.263 0.454 0.676
× 0.180 0.688 0.655
Isoeugenol 18.819/19.869 3.446 1.175 1.158 1.450 6.889
2.412 1.040 4.427 6.565
Homovanillic acid 23.810 0.915 × × 0.571 0.678
× × × 0.694
4-hydroxy-2-methoxycinnamaldehyde 25.380 1.746 1.105 0.507 0.193 2.145
1.540 0.484 2.537 0.596
4-acetyl-2-methoxyphenol 20.651 × × × × 1.349
× × 1.833 1.592
Vanillyl methyl ketone 21.554 0.327 × × 0.433 1.117
× × 0.952 0.994
Coniferyl alcohol 24.235/25.453 7.683 × 1.136 1.417 11.117
1.540 0.484 2.537 4.054

Note: the proportions of pyrolysed lignin compounds were calculated from their percentages in the total aromatic compounds. The data
on the top of each unit was from C. formosanus; the one at the bottom was from R. flavipes. † , no appearance in the pyrograms. The
structure of each compound is shown below:


C 2010 The Authors
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C Institute of Zoology, Chinese Academy of Sciences, Insect Science, 17, 277–290
286 J. Ke et al.
in termites; and further discuss the relationship between
wood-lignin modification and oxygen profiles along the
whole gut. The entire oxygen profiling pattern of the ter-
mite guts for both genera were consistent with the results
previously reported by Brune et al. (1995a) and Ebert &
Brune (1997). However, the detection of oxygen concen-
tration in the center of hindgut illustrates the high sensitiv-
ity of the microelectrode used in this study. Consumption
of oxygen in some parts of the gut may cause oxygen flux
from other sites, possibly leading to maintenance of the
anaerobic environment in other sites (Wertz & Breznak,
2007), and further resulting in varied oxygen distributions
across the entire gut. This idea supports Yang’s conclu-
sion (2005) that interactions of micro-organisms, and dif-
ferences in microenvironment inside and across habitat
boundaries, should influence the structure and diversity
of microbial communities within the ecosystem of the
termite gut. Lignin degraders might be one component
of the microflora that constitutes an oxygen sink in the
guts of WFTs. The steep oxygen gradients at the periph-
ery of the hindgut are maintained as a result of oxygen
flux to the midgut, or oxygen consumption by the in-
testinal microbiota (Brune, 1998), which occupy the bulk
of the hindgut volume (Breznak, 1984). Earlier reports
have indicated that WFTs prefer a diet containing lignin
over pure cellulose or fungus-infected wood, suggesting
the importance of lignin-modifying bio-factors in termite
guts (Kanai et al., 1982). Brune et al. (1995b) have stud-
ied the metabolism of monoaromatic model compounds
by termites and their gut microflora and concluded that
gut homogenates of the WFTs Nasutitermes lujae (Was-
mann) and R. flavipes mineralized lignin analogs only
if oxygen was present, which was consistent with the
oxygen requirement in other lignin-degrading systems.
In both termites examined in this study, the middle point
of the midgut exhibited higher oxygen concentration than
other gut regions, indicating a high possibility of lignin
oxidation there.
This work demonstrated both lignin modification and
gut microenvironments conducive to lignin modification
in WFTs. Early evidence of lignin degradation by termites
was obtained only from labeled heterogeneous biomass-
lignin or synthetic lignin (Cookson, 1987, 1988; Kyou
et al., 1996; Breznak & Brune, 1994; Itakura et al., 1995),
leading to ambiguity in the results. Chemical analysis of
lignin in WFTs’ food and feces have had varied results,
ranging from virtually minor changes (Katsumata et al.,
2007) to astonishingly high degradation values of 83%
(Lee & Wood, 1971). These earlier experiments charac-
terized only the Björkman lignin fraction in insect fe-
ces, limiting detection of modification of the entire lignin
sample. Uncertainty over whether termites attack lignin
Journal compilation 
C Institute of Zoology, Chinese Academy of Sciences, Insect Science, 17, 277–290
was reinforced by metagenome sequencing of the higher
termite species Nasutitermes corniger hindgut commu-
nity, in which no genes encoding known lignin-degrading
enzymes were found (Warnecke et al., 2007). However,
these findings are not in conflict with our hypothesis
that oxidation of lignin occurs mainly in the foregut and
midgut regions. In support of our hypothesis, tetram-
ethylammonium hydroxide (TMAH) thermochemolysis
analysis found significant alteration of the lignin poly-
mer of coniferous wood (softwood) after passage through
the gut of a lower WFT (Geib et al., 2008), including
propyl side-chain oxidation, ring demethylation and ring
hydroxylation.
Our work examined lignin modification in each seg-
ment of the termite gut. The results detail side-chain oxi-
dation/cleavage (depolymerization) and demethylation of
ring methoxyl groups of wood-lignin beginning in the
foregut, and continuing in the midgut. Ring demethyla-
tion makes the substrate more suitable for carbon-carbon
bond cleavage; with other reactions of side-chain oxida-
tion, ring opening will thus be facilitated (Goodell et al.,
2003), assuming guaiacols favor aromatic ring opening
and mineralization. Side-chain oxidation will cause de-
polymerization of lignin polymers or release of lignin
units from lignin–carbohydrate complex. Both of these
reactions are believed to require oxygen, as previous ex-
periments have demonstrated (Kedderis & Hollenberg,
1983; Ten Have et al., 2000). All currently known lignin-
degrading pathways are aerobic. Aerobic regions of ter-
mite guts, housing large populations of aerobic microbes
(Tholen, 1997; Brune et al., 1995a; Ohkuma, 2003) sup-
port the maintenance of anaerobic conditions in other
areas for fermentation of cellulose-derived monosaccha-
rides by termites (Adams & Boopathy, 2005; Johnson &
Barbehenn, 2000). The relatively high levels of oxygen
observed in the midgut were therefore consistent with
these reactions occurring in the midgut. Previous studies
have shown that, in fungi, phenol oxidase and peroxi-
dase are responsible for cleavage between the aromatic
and aliphatic portions of lignin, and phenolic and non-
phenolic oxidation of lignin (Janshekar & Fiechter, 1983;
Ten Have & Teunissen, 2001). Our findings support that
a similar system of catalysis might exist in the salivary
glands/foregut and midgut of WFTs (Tartar et al., 2009).
Furthermore, lignin modification should not only happen
in the foregut and midgut, but the hindgut might also par-
ticipate in the process of dealing with simpler products. As
lignin depolymerizes, it liberates phenolic groups, breaks
away from hemicellulose (Higuchi, 1990), and perhaps re-
leases low molecular weight lignin fragments (Janshekar
& Fiechter, 1983; Tartar et al., 2009), it will increase in
hydrophilia. It is speculated that when passing through the

C 2010 The Authors

hindgut, the easily digested byproducts, such as ester and
monomer lignin units, might be taken up through the gut
epithelium and oxidized aerobically by termite tissue or
gut symbionts; however, it needs further investigations to
clarify this. This process may be attributed to the function
of esterase and phenol oxidizing enzymes (Kirk et al.,
1980).
The presence of aromatic-degrading bacteria in termite
guts has been well documented (Watanabe et al., 2003;
Adams & Boopathy, 2005), indirectly supporting our ob-
servations of degradation of small plant aromatic com-
pounds in the hindguts of WFTs, which may contribute to
the carbon and energy requirement of the host. Pasti et al.
(1990) isolated 11 novel actinomycete strains with spe-
cific peroxidase activity from the guts of higher termites
(Termitidae). Later, Kuhnigk and König (1997) indicated
that the hindgut floras of xylophagous termites were able
to produce substrates for their hosts from dimeric lignin
model compounds in the presence of oxygen. The pres-
ence of bacteria that degrade monomer lignin and phenolic
compounds from decomposed wood lignin was reported
in the gut of Nasutitermes takasagoensis, Coptotermes
formosanus (Kinya et al., 1998; Harazono et al., 2003).
In addition, as demonstrated by Vu et al. (2004), there is
redox metabolism of iron in two WFTs; reduction of iron
(III) may provide redox potential for oxidation of lignin.
Since the epithelial regions of the gut are known to be
micro-oxygenic (Figs. 2, 3), iron (II) is likely subject to
rapid re-oxidation there (Vu et al., 2004), so that it can
again serve as a potent catalyst in the partial oxidation of
lignin fractions (Jamil & Hussain, 1992).
Although neither the exact rate of lignin degradation
by WFTs nor the specific micro-organisms or enzymes
responsible for lignin modification could be determined
at this time, their efficiency of wood disruption within
24 h is quite striking (Itakura et al., 1995; Katsumata
et al., 2007). In general, the mean retention time of di-
gesta in termite hindgut is estimated to be around 24–26 h
(Breznak, 1984). Our results agree with earlier predictions
regarding the distribution of phenol oxidase and esterase
along the gut of WFTs, as noted by Tartar et al. (2009) and
Wheeler et al. (2007) in the R. flavipes gut. Furthermore,
genes encoding peroxidases, which are also responsible
for wood-lignin disruption, have also been identified from
the R. flavipes gut (Tartar et al., 2009).
The results acquired in these studies have provided new
insights into our understanding of lignin degradation by
WFTs. Lignocellulosic degradation occurs throughout the
entire gut system, with initiation of lignin modification
before the hindgut. It is likely that WFTs have evolved op-
timal conditions and processes for efficient lignocellulose
degradation. Further understanding of the exact reactions

C 2010 The Authors
Journal compilation 
C Institute of Zoology, Chinese Academy of Sciences, Insect Science, 17, 277–290
Oxygen profiling and lignin modification in termite 287
and mechanisms that modify lignin in the termite gut will
contribute to our understanding of the biochemical roles
that these insects play in lignin modification, and pro-
mote industrial utilization of these processes for faster
and better access of enzymes to polymer carbohydrates.
Acknowledgments
This work was financially supported by the Agricultural
Research Center of Washington State University (WSU).
Dissolved oxygen microelectrode work supported by
Beyenal start up funds by WSU.
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Coptotermes formosanus Shiraki (Isoptera: Rhinotermitidae)
II. Selective defaunation of protozoa and its effect on cellulose
metabolism. Journal of the Japan Wood Research Society, 39,
227–230.
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lignin-derived compounds in anaerobic environments. Ge-
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Accepted March 1, 2010

C2010 The Authors
 Insect Science (2010) 17, 291–302, DOI 10.1111/j.1744-7917.2010.01346.x

ORIGINAL ARTICLE

Analysis of cellulolytic and hemicellulolytic enzyme activity

within the Tipula abdominalis (Diptera: Tipulidae) larval gut
and characterization of Crocebacterium ilecola gen. nov., sp.
nov., isolated from the Tipula abdominalis larval hindgut

Theresa E. Rogers and Joy Doran-Peterson

Microbiology Department, University of Georgia, Athens, Georgia, USA

Abstract In forested stream ecosystems of the north and eastern United States, larvae
of the aquatic crane fly Tipula abdominalis are important shredders of leaf litter detritus.
T. abdominalis larvae harbor a dense and diverse microbial community in their hindgut
that may aide in the degradation of lignocellulose. In this study, the activities of cellulolytic
and hemicellulolytic enzymes were demonstrated from hindgut extracts and from bacterial
isolates using model sugar substrates. One of the bacterial isolates was further characterized
as a member of the family Microbacteriaceae. Taxonomic position of the isolate within
this family was determined by a polyphasic approach, as is commonly employed for the
separation of genera within the family Microbacteriaceae. The bacterial isolate is Gram-
type positive, motile, non-sporulating, and rod-shaped. The G + C content of the DNA is
64.9 mol%. The cell wall contains B2γ type peptidoglycan, D- and L-diaminobutyric acid
as the diamino acid, and rhamnose as the predominant sugar. The predominant fatty acids
are 12-methyltetradecanoic acid (ai-C15:0 ) and 14-methylhexadecanoic acid (ai-C17:0 ). The
isolate forms a distinct lineage within the family Microbacteriaceae, as determined by
16S rRNA sequence analysis. We propose the name Crocebacterium ilecola gen. nov., sp.
nov., to accommodate this bacterial isolate. The type species is T202T (ATCC BAA-1359;
GenBank Accession DQ826511).
Key words cellulolytic, hemicellulolytic, hindgut

Introduction
Larvae of the aquatic crane fly Tipula abdominalis (Say)
(Diptera: Tipulidae) are major shredders of leaf litter de-
tritus in small order stream ecosystems of the northern
and eastern United States (Martin, 1987). The T. abdom-
inalis larval gut is composed of a foregut, gastric ceca,
midgut, pylorus, ileum, hindgut and rectum (Fig. 1). The
Correspondence: Joy Doran-Peterson, Microbiology Depart-
ment and Biofuels, Biopower, and Biomaterials Initiative, Uni-
versity of Georgia, 527 Biological Sciences Building, 1000
Cedar Street, Athens, GA 30602, USA. Tel: 706 542 4115; fax:
706 542 2674; email: jpeterso@uga.edu
pH of the midgut ranges from 8.5 to 11.6, with the high-
est alkalinity located in the middle of the midgut, and
the hindgut pH range is from 7.1 to 7.5 (Martin, 1987).
The midgut and the hindgut harbor a dense microbial
community, while other sections of the alimentary tract
are not colonized. The midgut lumen consists of a mi-
crobial community similar to that found on conditioned
leaf detritus with 108 cells/mg, and the midgut wall is not
colonized. The hindgut lumen and wall are densely colo-
nized, with 109 and 1010 cells/mg, respectively (Klug &
Kotarski, 1980). The hindgut is compartmentalized into
anterior and posterior sections, and the anterior hindgut
is commonly referred to as the fermentation chamber
(Martin, 1987). The morphological diversity of the
microbial community residing in the T. abdominalis


C 2010 The Authors 291
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C Institute of Zoology, Chinese Academy of Sciences
292 T. E. Rogers & J. Doran-Peterson
Fig. 1 Tipula abdominalis alimentary tract. Figure is redrawn
from Klug and Kotarski (1980). Figure is not drawn to scale.
larval alimentary tract was studied via microscopy and
direct isolation (Klug & Kotarski, 1980). The microbial
communities associated with the anterior and posterior
hindgut lumen and wall are different and more morpho-
logically diverse than the microbial community associated
with leaf detritus. The posterior hindgut wall harbors the
most diverse community overall. Flagellate and ciliate
protozoans are not common inhabitants of the T. abdomi-
nalis larval gut, and were only found in young or unhealthy
larvae (Klug & Kotarski, 1980).
The majority of T. abdominalis larval nutrition is de-
rived from leaf litter detritus rather than microbial biomass
(Cummins & Klug, 1979). It is unlikely that degradation
of cellulosic material is due to ingested fungal cellulases
since these enzymes would be inactivated by the high
pH of the midgut (Martin, 1987; Suberkropp & Klug,
1974). Sinsabaugh and colleagues successfully demon-
strated exoglucanase activity within the hindgut and the
assimilation of purified cellulose by transport of organic
and amino acids across the gut wall (Sinsabaugh et al.,
1985). These data lead to the hypothesis that the microbial
symbionts are responsible for the degradation of cellulosic
material in the T. abdominalis larval gut rather than insect
tissue-level synthesized cellulases. However, this contri-
bution of the gut microbiota to the degradation of leaf
litter detritus within stream ecosystems has not yet been
fully established.
This study aimed to demonstrate the activity of cellu-
lolytic and hemicellulolytic enzymes within the T. abdom-
inalis larval gut. The midgut and hindgut of T. abdomi-
Journal compilation 
C Institute of Zoology, Chinese Academy of Sciences, Insect Science, 17, 291–302
nalis were separated in order to assay their activities at
physiologically relevant pH values of 11.0 and 7.4, re-
spectively (Martin et al., 1980). Although the high pH
in the midgut is generally associated with proteolytic ac-
tivity (Graca & Barlocher, 1998) and inactivation of fun-
gal cellulases (Barlocher & Porter, 1986; Martin et al.,
1980; Sinsabaugh et al., 1985; Suberkropp et al., 1976),
alkaline cellulases from bacterial soil isolates have been
described previously (Singh et al., 2004). It was therefore
necessary to measure cellulolytic and hemicellulolytic ac-
tivity within the midgut at pH 11.0. Previous studies of
the bacterial isolates from T. abdominalis hindgut ho-
mogenates demonstrated activity on methylumbelliferyl-
(MU-) conjugated sugars (J. Doran-Peterson, submitted).
MU-conjugated substrates have also been used to demon-
strate the activity of extracellular enzymes in marine sys-
tems, the digestive tracts of freshwater snails, and of the
larvae of two Trichoptera species (Brendelberger, 1997;
Hoppe, 1983; Schulte et al., 2003). In this study, MU-
conjugated sugars were used to measure cellulolytic and
hemicellulolytic enzyme activity from T. abdominalis lar-
val gut extracts.
In addition to demonstrating the activity of cellulolytic
and hemicellulolytic enzymes within the T. abdominalis
larval gut, we also report the isolation of strain T202T
from the hindgut of a T. abdominalis larva. Characteriza-
tion was performed using a polyphasic approach, which
differentiates taxonomic lineages by assessing both phy-
logenetic and phenotypic characteristics. Based on the
phylogenetic and chemotaxonomic data presented within,
we propose a novel genus within the family Microbacteri-
aceae, “Crocebacterium” gen. nov., and the type species,
“Crocebacterium ilecola” sp. nov., to accommodate this
bacterium.
Materials and methods
Collection site
Fourth instar T. abdominalis larvae were collected from
Shope Fork Creek, Coweeta, NC. Stream parameters were
as follows: pH 5.35; dissolved oxygen, 11.2 mg/L; water
temperature, 7.7◦ C; conductivity, 14.58 μs/cm; turbidity,
0.47 nephelometric turbidity units; alkalinity, 12.0 mg/L;
< 1 mg/L NO3 − ; and < 0.1 mg/L PO4 3− . Larvae were
stored at 12◦ C in an aerated 4 L container filled with
filter-sterilized stream water and leaves collected from
the stream of capture. Water was changed every 3–4 days.
Of the 12 larvae, four per day were dissected and their gut
homogenates were assayed 14, 23 and 28 days following
capture.

C 2010 The Authors

Larval dissection
Tipula abdominalis larval midguts (with foregut at-
tached) and hindguts were removed and separated dur-
ing dissection under : buffered salts solution (BSS)7.4
buffer, pH 7.4 (10.8 mmol/L K2 HPO4 , 6.9 mmol/L
KH2 PO4 , 21.5 mmol/L KCl, 24.5 mmol/L NaCl,
1.0 mmol/L DTT) (Leadbetter & Breznak, 1996) (Fig. 1).
Midguts were placed in 5 mL BSS11 buffer, pH 11.0
(25 mmol/L Na2 CO3 , 24.5 mmol/L KCl, 1.0 mmol/L
DTT), and hindguts were placed in 5 mL BSS7.4 buffer,
pH 7.4.
Sample preparation
Protocol was modified from Schulte et al. (2003) and
Brendelberger (1997). Midguts and hindguts were soni-
cated in a Branson 1510 ultrasonic bath (Branson Ultra-
sonics, Danbury, CT, US) for 1 min, then centrifuged at
3 500 g and 4◦ C for 30 min (Schulte et al., 2003). The
supernatant was removed and placed into a new tube, and
from here on is termed the “enzyme extract.” The cen-
trifuged gut pellet was resuspended in buffer of appropri-
ate pH and from here on is termed the “resuspended gut
extract.” From both the enzyme extract and resuspended
gut extract, a 1 mL aliquot was filtered through a 0.2 μm
filter to screen for cell-free enzyme activity: “filtered en-
zyme extract” and “filtered resuspended gut extract.” An-
other 1 mL aliquot was boiled for 30 min to serve as a
negative control: “boiled enzyme extract” and “boiled re-
suspended gut extract” (Schulte et al., 2003). To determine
if controls were free of viable micro-organisms, 200 μL
aliquots of the filtered and boiled extracts were spread-
plated onto tryptic soy agar (TSA, DIFCO, Detroit, MI,
US) and compared to plated enzyme extracts and resus-
pended gut extracts, described below. All gut extracts were
assayed on the day of dissection. A previously studied
bacterial isolate from the T. abdominalis gut (isolate 27,
accession number AY504453) known to demonstrate ac-
tivity on all MU-substrates in this study was used as a pos-
itive control. The unfiltered gut extracts represent micro-
organisms residing within the gut lumen and those that
were easily dislodged from the gut wall. The resuspended
gut extracts represent the micro-organisms attached to the
gut wall, and the filtered gut extracts represent cell-free
enzymes.
Preparation of stock solutions and standards
Stock solutions of the MU-standard and substrates
were prepared by dissolving in 5 mL of 70% (v/v)

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C Institute of Zoology, Chinese Academy of Sciences, Insect Science, 17, 291–302
Enzyme activity within Tipula abdominalis 293
ethanol, then diluting to 50 mL with autoclaved dis-
tilled water to a final concentration of 2 mmol/L.
Standard concentrations of methylumbelliferone sodium
salt (Sigma, St Louis, MO, US) were 100.0, 50.0,
25.0, 20.0, 15.0, 10.0 and 1.0 μmol/L (Schulte et al.,
2003). New standards were prepared daily since flu-
orescence diminished upon storage at −20◦ C. The
five MU-conjugated sugars used as model substrates
were 4-methylumbelliferyl-α-L-arabinofuranoside
(MUA, Sigma), 4-methylumbelliferyl-β-D-cellobioside
(MUC, Sigma), 4-methylumbelliferyl-1 β-D-
glucopyranoside (MUG, Sigma), 4-methylumbelliferyl-
β-D-mannopyranoside (MUM, Sigma), and 4-
methylumbelliferyl-β-D-xyloside (MUX, Sigma).
In contrast to the methylumbelliferone sodium salt
standard, the MU-conjugated substrate solutions were
stable upon storage at −20◦ C.
Enzyme assay
Enzyme activity was assayed in black 350 μL 96-well
plates (NUNC, Rosklide, Denmark) with a spectroflu-
orometer Spectra MAX R
GEMINI EM (Molecular De-
vices, Sunnyvale, CA, US). Unfiltered, filtered, and boiled
enzyme extracts and resuspended gut extracts (40 μL)
were mixed with BSS buffer, pH 11.0 for midgut and
7.4 for hindgut (155 μL), and MU-substrate (5 μL). Top
read, endpoint fluorescence enzyme assays began with
substrate addition and were incubated for 20–30 min at
25◦ C. Fluorescence was measured at 360 nm emission and
435 nm excitation with no cutoff wavelength, and values
were the average of 20 readings (Schulte et al., 2003).
Fluorescence values directly relate to liberated methy-
lumbelliferone concentrations.
Isolation and screening of hindgut microbiota
Hindgut and resuspended gut extracts of two larvae
were plated onto TSA and Luria-Bertani agar (LBA,
DIFCO) media in 50 and 75 μL aliquots and incubated at
room temperature until no new colonies appeared (after
9 days). A total of 198 colonies were streaked onto ad-
ditional TSA or LBA plates for isolation. Colonies were
picked according to morphology and incubation time be-
fore colony appearance to reduce repetition of isolates,
picking only 3–4 colonies with similar morphology if
found on different plates or from different gut extracts.
Glycerol stocks of 40% (w/v) glycerol were made of each
bacterial isolate for storage at −80◦ C. Isolates were then
screened on TSA + 1 g/L MUA, MUC, MUG, MUM
or MUX by replica plating. Cleavage of MU-conjugated
294 T. E. Rogers & J. Doran-Peterson

sugars, MUA, MUC, MUG, MUM and MUX was deter- tion was performed by gas chromatography (MacKenzie,
mined by the method of Sharrock (1988). Fluorescence 1987). Sugars present in the cell wall were determined
was detected by exposure to UV light after incubation for as described by Staneck and Roberts (1974). Fatty acid
1 week. methylesterase (FAME) analysis was performed by MIDI
LABS (Newark, DE, US).
Morphological, physiological and chemotaxonomic
characterization of isolate T202T
DNA extraction and phylogenetic analysis of isolate
T T202T
Isolate T202 colony morphology was observed on
TSA at 24–25◦ C. Cell morphology was observed by
DNA extraction was modified from Harwood and
phase-contrast microscopy and bright-field microscopy.
Cutting (1990). Briefly, pelleted cells were resuspended
Gram-staining was performed as described (Ayers, 2000).
in lysis buffer (50 mol/L Na2 EDTA, 0.1 mol/L NaCl,
Motility was studied by phase-contrast microscopy and
pH 7.5) with 3 mg lysozyme/mL and incubated at 37◦ C
motility test medium (BBL) (2003b). Temperature range
for 10 min. Sodium dodecyl sulfate (SDS) was added to
and the optimum for growth were determined in TSB, pH
1.5% (w/v) final concentration, and the sample was in-
7.0. For temperatures above 5◦ C, a temperature gradient
cubated an additional 5 min at 37◦ C. Phenol (pH 7.8)
incubator (Scientific Industries, Bohemia, NY, US) was
extraction, chloroform : isoamyl alcohol (24 : 1) extrac-
used with agitation. For temperatures between 0◦ C and
tion, and ethanol precipitation were then performed. DNA
5◦ C, cells were grown on TSA, pH 7.0. pH range and the
G + C content was determined as described in Mesbah
optimum for growth were determined in TSB at 24–25◦ C.
and Whitman (1989) and confirmed by the DSMZ by the
NaCl tolerance was determined on LBA with NaCl con-
methods of Cashion et al. (1977), Mesbah and Whitman
centrations between 0% and 10% (w/v). Growth under
(1989), Tamaoka and Komagata (1984) and Visuvanathan
anaerobic and microaerobic conditions was examined us-
et al. (1989). Sequencing of the full 16S rRNA gene
ing BBL GasPak R
jar systems (BBL Microbiology Sys-
was performed by MIDI LABS (Newark, DE, US) us-
tems, Cockeysville, MD, US). Presence of endospores was
ing primers corresponding to Escherichia coli 16S rRNA
determined by staining as previously described (Doetsch,
positions 005 and 1540. Sequences with high similarity
1981).
to isolate T202T were obtained from a BLAST search
Isolate T202T was grown at 24–25◦ C for all physio-
with GenBank (http://www.ncbi.nlm.nih.gov). The 16S
logical tests mentioned below. Catalase activity was de-
rRNA gene sequence from isolate T202T was aligned with
termined by the addition of 3% (v/v) hydrogen peroxide
the multiple alignment program ClustalX (Thompson
solution. Oxidase activity was tested using freshly pre-
et al., 1997) against previously determined Microbacteri-
pared 1% tetramethyl-p-phenylenediamine dihydrochlo-
aceae sequences obtained from the public database NCBI
ride (Tarrand & Groschel, 1982). Methyl red (MR),
(http://www.ncbi.nlm.nih.gov), then edited with GeneDoc
Voges-Proskauer (VP), nitrate reduction, formation of
(Nicholas et al., 1997). Phylogenetic analyses were per-
H2 S, urease activity, extracellular DNase activity, and
formed with the program Mega2 (Mukhopadhyay et al.,
hydrolysis of casein and starch were determined follow-
2005). To determine consistency between analysis meth-
ing the manufacturer’s instructions (DIFCO). Hydroly-
ods, phlyogenetic distance trees were generated using the
sis of carboxymethyl cellulose (CMC), xylan, and pectin
Kimura two-parameter and Jukes-Cantor models and the
was determined as previously described (MacFaddin,
neighbor-joining, minimum evolution, and maximum par-
1985; Mondou et al., 1986; Vera & Dumoff, 1974; Wood
simony algorithms (Kimura, 1980; Saitou & Imanishi,
& Kellogg, 1988). Various enzyme activities and acid
1989).
production from a variety of carbohydrates were inves-
tigated using API Staph, Strep, and Coryne systems
(BioMérieux, Marcy l’Etoile, France) following the man- Results
ufacturer’s instructions, except incubation was at 25◦ C
for 48 h. Antibiotic tolerance was determined by the Enzyme assay
Kirby-Bauer disc-diffusion method using BBLTM Sensi-
Discs (BBL Microbiology Systems) (2003a). Cell wall All five MU-conjugated substrates were hydrolyzed
analysis was performed by the DSMZ identification ser- by the unfiltered and filtered hindgut enzyme extracts
vice (Braunschweig, Germany). Peptidoglycan structure (Figs. 2 and 3). Hydrolysis of MUG was up to 13
was analyzed as described by Schleifer and Kandler times greater than hydrolysis of the other four MU-
(1972) and Schleifer (1985). Amino acid quantifica- conjugated sugars by hindgut enzyme extracts. Both


C 2010 The Authors
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C Institute of Zoology, Chinese Academy of Sciences, Insect Science, 17, 291–302
 Enzyme activity within Tipula abdominalis 295

Fig. 2 MUG hydrolyzed (μmol/L/g dry gut weight) by 10 individual Tipula abdominalis larvae hindgut enzyme extracts and resuspended
guts. Hindgut enzyme extracts and resuspended guts are presented as unfiltered (grey), filtered (black) and boiled (white). Hindgut
enzyme extracts from animals 1 and 2, 3–6 and 7–10 were assayed 14, 23 and 28 days after capture, respectively. Resuspended guts
from animals 1–3, 4–6 and 7–10 were assayed 14, 23 and 28 days following capture, respectively. All assays were performed between
20 and 30 min.

unfiltered and filtered resuspended hindgut extracts hy-
drolyzed MUG, MUA and MUX, while only unfil-
tered resuspended hindgut extracts hydrolyzed MUC
significantly more than the negative controls (Figs. 2
and 3). Activity of unfiltered and filtered resuspended
hindgut extracts on MUM was similar to negative con-
trols (Fig. 3). All unfiltered and filtered midgut enzyme
extracts and resuspended midgut extracts had similar ac-
tivity to the negative controls (data not shown). No bac-
terial colonies formed on TSA plates inoculated with
filtered or boiled extracts. All enzyme extracts and re-
suspended gut extracts were assayed the day of dissec-
tion since significant enzyme activity was lost when
frozen at −20◦ C overnight and assayed the following
day.
Isolation and screening of hindgut microbiota
Of the 198 isolates, 35.3, 39.4, 58.1, 27.3 and 35.9% dis-
played activity on MUA, MUC, MUG, MUM and MUX,
respectively. These numbers are similar to the relative
hindgut enzyme extract activity on each of the five MU-
conjugated sugar substrates (Figs. 2 and 3). For example,
enzyme activity was highest with MUG and lowest with
MUM.
Morphological, physiological and chemotaxonomic
characterization of isolate T202T
Isolate T202T colonies were round, smooth, shiny, con-
vex, opaque, yellow when grown in the light, and white

Fig. 3 Average Tipula abdominalis hindgut enzyme extract and resuspended hindgut activity (μmol/L/g dry gut weight) on four
methylumbelliferyl- (MU)-substrates. Hindgut enzyme extracts and resuspended guts are presented as unfiltered (grey), filtered (black)
and boiled (white). Data are for 10 animals and error bars indicate standard deviations. All unfiltered and filtered enzyme extracts
and resuspended guts are significantly different (P ≤ 0.001), determined by Student’s t-tests, from boiled enzyme extracts except
unfiltered resuspended gut activity on methylumbelliferyl-β-D-mannopyranoside (MUM) and filtered resuspended gut activity on
4-methylumbelliferyl-β-D-cellobioside (MUC) and MUM. All assays were performed between 20 and 30 min.


C 2010 The Authors
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C Institute of Zoology, Chinese Academy of Sciences, Insect Science, 17, 291–302
296 T. E. Rogers & J. Doran-Peterson
when grown in the dark. Long rods were the predominant
morphology in young cultures (width 0.7–1.5 μm, length
3–4.5 μm, 18 h), and short rods predominate older cul-
tures (width 0.4–0.7 μm, length 0.7–1.5 μm, 50 h), yet
a marked rod-coccus cycle was not observed. When cells
were longer, they often occurred in short chains of 2–4
cells, each at a slight angle to the other at the point of
attachment. Gram stain was negative for a young culture
(18 h), and positive for an older culture (50 h). Motility
was greater in younger cultures than older cultures (18 h
and 50 h, respectively). Optimal cell growth was at 21–
25◦ C, pH 7.0, and 0.0–0.5% (w/v) NaCl. Weak growth
occurred at 0◦ C and 37◦ C, pH 10 and 2.5% (w/v) NaCl.
No growth occurred at 42◦ C, pH 5 and 5% (w/v) NaCl.
Cells grew best aerobically, and less growth occurred un-
der microaerobic conditions. Growth was not detected
after a 4-week incubation under anaerobic conditions, but
growth occurred when transferred to aerobic conditions.
Cells did not form endospores. A long-to-short rod cy-
cle, variable Gram stain, lack of sporulation, microaero-
bic growth, and minimum growth temperatures near 0◦ C
are characteristics found among many members of the
Microbacteriaceae family (Groth et al., 1996; Kampfer
et al., 2000; Mannisto et al., 2000; Sheridan et al., 2003;
Zlamala et al., 2002).
Catalase and oxidase tests were positive. Nitrate was
reduced to NH4 + . MR and VP tests were negative,
and isolate T202T did not form H2 S. No hydrolysis
of hippurate, gelatin, casein, starch, CMC, xylan or
pectin occurred. Isolate T202T tested positive for the en-
zyme activities of alkaline phosphatase, β-glucosidase,
α-glucosidase, β-glucuronidase, β-galactosidase, and
urease. T202T did not produce α-methyl-D-glucosidase,
N-acetyl-glucosaminase, α-galactosidase, leucine ary-
lamidase, citrase or pyrrolidonyl arylamidase. Under aer-
obic conditions, acid was produced from D-glucose, D-
fructose, D-mannose, D-maltose, lactose, D-trehalose,
D-mannitol, xylitol, D-melibiose, raffinose, xylose and
sucrose. Growth did not occur under anaerobic condi-
tions, therefore acid production from carbohydrates un-
der anaerobic conditions was not detected. Degradation
of MUA, MUC, MUG and MUX occurred. MUM was
not degraded. Isolate T202T was resistant to kanamycin
(30 μg) and penicillin (10 μg), but not to ampicillin
(10 μg), ciprofloxacin (5 μg), erythromycin (15 μg),
rifampin (5 μg), polymyxin B (300 μg), streptomycin
(10 μg), trimethoprim (5 μg) or vancomycin (30 μg).
The cell wall peptidoglycan was of the type B2γ .
The cell wall contained alanine, glycine, D- and L-
diaminobutyric acid (DAB), and glutamic acid in the mo-
lar ratio 0.6 : 1.0 : 2.5 : 1.0. The predominant sugar
in the cell wall was rhamnose, and the minor sugar
was mannose. No mycolic acids were present. The most
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C Institute of Zoology, Chinese Academy of Sciences, Insect Science, 17, 291–302
abundant lipids were 12-methyltetradecanoic acid (ai-
C15:0 ; 63.5%) and 14-methylhexadecanoic acid (ai-C17:0 ;
21.0%). Other lipids present in significant amounts were
13-methyltetradecanoic acid (i-C15:0 ; 1.35%), hexade-
canoic acid (C16:0 ; 5.09%) and 14-methylpentadecanoic
acid (i-C16:0 ; 7.03%). Tetradecanoic acid (C14:0 ), 12-
methyltridecanoic acid (i-C14:0 ), pentadecanoic acid
(C15:0 ), 15-methylhexadecanoic acid (i-C17:0 ), octade-
canoic acid (C18:0 ), and cyclohexyl acid w9c-18:1 were
each present as < 1%.
Phylogenetic analysis of isolate T202T
The G + C content of the DNA was 64.9 mol%. Isolate
T202T full 16S rRNA gene sequence (1526 nucleotides)
had 96.5% identity to an unidentified actinobacterium
(partial sequence) cultured from African millipede fecal
pellets. Both an uncultured actinobacterium from an in-
dustrial biofilter (partial sequence) (Friedrich et al., 2002)
and a Leifsonia poae isolate from nematode galls (partial
sequence) (Evtushenko et al., 2000) had 96.2% sequence
identity to isolate T202T 16S rDNA.
Results from phylogenetic distance analyses were sim-
ilar using the Kimura two-parameter or Jukes-Cantor
model and the neighbor-joining, minimum evolution, or
maximum parsimony algorithm. Phylogenetic trees based
on these distance analyses show that isolate T202T is
closely related to the genera Agreia, Frigoribacter, Sub-
tercola, Rhodoglobus and Salinibacterium, yet forms a
distinct lineage among these genera within the Microbac-
teriaceae family (Fig. 4). Isolate T202T is shown to clus-
ter with the genus Frigoribacterium, but the low boot-
strap value (Saitou & Imanishi, 1989) indicates that this
placement is uncertain. Other characteristics separating
isolate T202T from other genera within this cluster are
the cell wall amino and diamino acids, cell wall sug-
ars, fatty acid profile, and menaquinone composition
(Table 1). Most notably different are the fatty acids with
ai-C15:0 composing over 60% of the total fatty acids. The
genus Rhodoglobus has the closest fatty acid composition
with 53.2% ai-C15:0 and 22.6% ai-C17:0 , but this genus has
18.8% i-C16:0 compared to 7.0% for isolate T202T . Also,
the genus Rhodoglobus has D-ornithine as the diamino
acid within the cell wall, whereas isolate T202T has D-
and L-DAB (Table 1).
Discussion
Cellulose and hemicellulose degradation
Cellulase and hemicellulase activity have been success-
fully demonstrated in hindgut extracts of T. abdominalis

C 2010 The Authors
 Enzyme activity within Tipula abdominalis 297

Fig. 4 Phylogenetic tree based on 16S rRNA gene sequences of Crocebacterium ilecola gen. nov., sp. nov. and genera of the family
Microbacteriaceae. Accession numbers for nucleotide sequences are in parentheses. The numbers on the tree indicate bootstrap values,
expressed as a percentage of 500 replicates. Only bootstrap values of 50% or greater are shown. Bar indicates 1 nucleotide substitution
per 100 nucleotides. Brevibacter linens (DSM 20425T , X77451) served as an out-group sequence to estimate the root position of the
tree.

larvae by use of MU-conjugated model substrates. The
MU-conjugated sugars model the ends of large poly-
meric sugars or oligosaccharides. Cleavage of these sub-
strates infers the ability to degrade sugar polymer ends
or oligosaccharides produced by the partial degradation
of lignocellulosic material. No cellulolytic or hemicel-
lulolytic activity was detected within the midgut, which
is not surprising because the pH of the midgut suggests
proteolytic, not cellulolytic or hemicellulolytic activity.
Enzyme activity profiles were similar for animals in
captivity for 14, 23 and 28 days, therefore demonstrating
that captivity between days 14 and 28 had a minimal affect
on the detection of hydrolytic enzyme capabilities. Larvae
were not assayed on the day of capture; therefore it cannot
be determined if a change in hydrolytic enzyme activity
occurred between days 0 and 14. It is probable that as
food sources change, a shift will occur in the microbial
community and/or enzyme production to allow for better
degradation of the new food source. In this study, larvae
were fed leaves collected from the stream of capture to
reduce a potential shift in the microbial community or
enzyme production.


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298 T. E. Rogers & J. Doran-Peterson

Table 1 Distinguishing characteristics of the genera Agreia, Frigoribacterium, Rhodoglobus, Salinibacterium, Subtercola and isolate
T202T .

Characteristic† Isolate T202T Agreia‡ Frigoribacterium Rhodoglobus Salinibacterium Subtercola

Peptidoglycan type B2γ B B2β B2α B B2γ

Cell wall diamino acids D, L-DAB L-DAB, D-Orn D-Lys D-Orn D-Lys, D-Orn DAB
Other cell wall amino acids Ala, Gly, Glu Ala, Gly, Hyg or Glu Ala, Gly, Hsr Ala, Gly, Glu Ala, Gly, Glu ?Hyg
Cell wall sugars
major Rhamnose Rhamnose nr nr nr Rhamnose, Xylose
minor Mannose Mannose and fucose nr nr nr Mannose, Glu, Rib
Major fatty acids (%)
ai-C17:0 21.0 14.4–22.1 7.0–14.8 22.6 2.8 3.5–6.8
ai-C15:0 63.5 46.7–48.0 37.8–48.3 53.2 40.4 46.1–51.6
i-C16:0 7.0 17.2–30.1 10.0–16.7 18.8 34.7 4.2–10.2
i-C15:0 1.3 ≤ 1.0 1.3–1.8 0.79 6.6 < 1.0–4.3
i-C14:0 < 1.0 < 1.0 ≤ 1.0 1.1 14.7 < 1.0–6.7
C16:0 5.1 2.7–5.2 4.4–26.5 0.55 – –
Major menaquinones nr 10 9 11, 12 11 9, 10
G+ C content (mol%) 65 65–67 71.1 62 61 64–68
Temperature optima (◦ C) 21–25 24–26 4–8 18 25–28 15–17
Temperature range (◦ C) 1–37 < 37 −2–28 −2–21 4–37 −2–28
Motility + + + nr – –
Colony color Y, W Y, O Y R nr Y
†
Peptidoglycan type as designated by Schleifer and Kandler (1972).
‡
Data for known genera were collected from Behrendt et al. (2002), Evtushenko et al. (2001), Kyun Han et al. (2003), Mannisto et al.
(2000), Sheridan et al. (2003) and Schumann et al. (2003). DAB, diaminobutyric acid; Lys, lysine; Orn, ornithine; Ala, alanine; Gly,
glycine; Glu, glutimate; Hsr, homoserine; Hyg, hydroxyglutamate; ai, anteiso-branched fatty acids; i, iso-branched fatty acids; nr, not
reported; R, Red; W, white; Y, yellow.

Fermentation of the cellulolytic larval diet is expected
within the hindgut since it possesses an enlarged anterior
paunch, which is an analogous structure to the termite
paunch where fermentation is facilitated by retarding the
passage of food through the hindgut (Klug & Kotarski,
1980; Martin, 1987). Also, both the degradation of cel-
lulose to organic acids and the transport of acetate into
the hemolymph as a carbon and energy source for T. ab-
dominalis have been demonstrated (Lawson et al., 1984;
Sinsabaugh et al., 1985). An analysis of enzyme activity
from the anterior paunch verses the posterior hindgut is
needed to determine where the majority of the cellulose
degradation occurs.
Cellulase and hemicellulase activity from hindgut
microbiota
Many of the microbial isolates from the larval hindgut
demonstrated the ability to degrade cellulosic and/or
hemicellulosic material, indicating a possible source of
cellulolytic and/or hemicellulolytic enzymes. Isolation of
gut-associated microbiota was performed under aerobic
conditions on rich media (TSA and LBA). No special
efforts were made to enrich for cellulolytic or hemicel-
lulolytic bacteria or to isolate anaerobes because the cul-
turing techniques were originally designed for compar-
ing the quantity of bacteria in the enzyme extracts and
resuspended gut extracts to the boiled and filtered con-
trols. It was only after isolation that the magnitude of
morphological diversity found by using readily available
media, rather than enrichments or specialized media, was
realized. Other studies from this research group have cul-
tured bacteria from various areas of the T. abdominalis
alimentary tract with various types of media under both
aerobic and anaerobic conditions (J. Doran-Peterson, un-
published data).
Taxonomic conclusions of isolate T202T
Isolate T202T was fully characterized because of its
ability to degrade four of the five MU-conjugated sub-
strates tested. Although isolate T202T has up to 96.2%


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sequence identity to other genera within the family
Microbacteriaceae, this high percent identity is common
between members of other genera within this family. For
example, the 16S rRNA sequence identities of the genera
Agreia and Subtercola are 96.8%–97.1%, and identities
for Frigoribacterium, Rathayibacter, and Clavibacter are
96.1%–97.1% (Evtushenko et al., 2001; Kampfer et al.,
2000). These were characterized as separate genera based
on polyphasic taxonomic studies rather than 16S rRNA
sequence similarity alone. Of the phenotypic properties
studied, chemotaxanomic characteristics, such as pepti-
doglycan composition, and fatty acid profile, were impor-
tant distinguishing characteristics between these genera
(Evtushenko et al., 2002; Tsukamoto et al., 2001). Con-
sidering both chemotaxanomic characteristics and 16S
rRNA analysis, isolate T202T clearly forms a separate
genus within the family Microbacteriaceae. We propose
a new genus within the Microbacteriaceae family, Cro-
cebacterium gen. nov., and the type species, Crocebac-
terium ilecola sp. nov., to accommodate this bacterial
isolate.
Ecological implications of isolate T202T
Many members of the Microbacteriaceae family are
associated with plants (Behrendt et al., 2002; Dorofeeva
et al., 2002, 2003; Evtushenko et al., 2000, 2001, 2002;
Sasaki et al., 1998). The source of inoculum for the T. ab-
dominalis hindgut is believed to be ingested leaf detritus
(Klug & Kotarski, 1980; Lawson et al., 1984); therefore
it is not surprising to find members of the Microbacte-
riaceae family within this gut system. Additionally, this
bacterium was most likely a gut wall-associated bacterium
since it was isolated from the pelleted hindgut (Dillon &
Dillon, 2004). Most gut wall-associated bacteria are con-
sidered resident rather than transient bacteria because they
can resist the flow of material through the gut by at-
tachment or inhabiting small crevices along the gut wall.
Because these bacteria are constant inhabitants of the
hindgut, they may have a specific role within the mi-
crobial community. One possible role is the degradation
of cellulosic material in the T. abdominalis larval gut (Dil-
lon & Dillon, 2004; Sinsabaugh et al., 1985). Although
isolate T202T was unable to hydrolyze large polymeric
carbohydrates such as starch, CMC and xylan, it was able
to cleave four of the five MU-conjugated sugars tested.
If isolate T202T is a resident of the hindgut, it may oc-
cupy a niche within the microbial community degrading
oligosaccharides produced by the partial degradation of
lignocellulosic material. To determine whether this bac-
terial isolate, or any other isolate from the T. abdominalis
hindgut, is a resident rather than a transient bacterium,

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Enzyme activity within Tipula abdominalis 299
studies such as fluorescent in situ hybridization may be
employed.
Description of Crocebacterium gen. nov.
Crocebacterium (Cro.ce.bac.ter.ium. L. adj. croceus
saffron-colored, golden, yellow; Gr. n. bakterion small
rod; Crocebacterium a small yellow bacterium).
Cells are microaerobic to aerobic, Gram-type positive,
high G + C, motile, non-spore forming, irregular rods.
Colonies are round, smooth, shiny, convex, opaque and
may produce pigments. Growth occurs at mesophilic tem-
peratures and neutral pH. The cell wall peptidoglycan is
of the type B2γ , and the diamino acid is DAB (D- and L-
forms). No mycolic acids are present. Predominant fatty
acids are 12-methyltetradecanoic acid (ai-C15:0 ) and 14-
methylhexadecanoic acid (ai-C17:0 ). The type species is
Crocebacterium ilecolaT .
Description of Crocebacterium ilecola sp. nov.
Crocebacterium ilecola (i.le.co.la. L. n. ile gut; Gr. adj.
–cola inhabitant; ilecola inhabitant of the gut).
In addition to those characteristics described for the
genus, Crocebacterium ilecola does not have a marked
rod–coccus cycle. Cells of younger cultures are motile,
long rods, and stain Gram-negative, and cells of older
cultures are less motile, shorter rods, and stain Gram-
positive. Colonies appear yellow when grown in the light
and white when grown in the dark. The DNA G + C
content is approximately 65 mol%. Cell wall amino acids
are alanine, glycine, D- and L-DAB, and glutamic acid
(0.6 : 1.0 : 2.5 : 1.0 molar ratio). Cell wall sugars are rham-
nose (major) and mannose. Temperature range for growth
is 0–37◦ C, optimal growth occurs between 21–25◦ C, and
no growth occurs at 42◦ C. Growth occurs at a pH range of
6–10, optimal growth occurs at pH 7.0 and no growth oc-
curs at pH 5.0. Range of NaCl concentrations for growth is
0–2.5% (w/v), optimal growth occurs between 0.0–0.5%
(w/v) NaCl, and growth is inhibited at 5.0% (w/v) NaCl.
Catalase and oxidase tests were positive. Nitrate was re-
duced to NH4 + . MR and VP tests were negative. Activi-
ties were positive for alkaline phosphatase, β-glucosidase,
α-glucosidase, β-glucuronidase, β-galactosidase and
urease. Under aerobic conditions, acid is formed from D-
glucose, D-fructose, D-mannose, D-maltose, lactose, D-
trehalose, D-mannitol, xylitol, D-melibiose, raffinose, xy-
lose or sucrose. Hippurate, gelatin, casein, starch, CMC,
xylan and pectin are not hydrolyzed. Resistance was ob-
served to kanamycin (30 μg) and penicillin (10 μg), but
susceptible to ampicillin (10 μg), ciprofloxacin (5 μg),
erythromycin (15 μg), rifampin (5 μg), polymyxin B
300 T. E. Rogers & J. Doran-Peterson
(300 μg), streptomycin (10 μg), trimethoprim (5 μg) and
vancomycin (30 μg).
Acknowledgments
We wish to thank Drs. Barny Whitman, Juergen Wiegel
and Ellen Neidle for critical review of the manuscript. The
authors also thank Drs. Barny Whitman and Yong Jin Lee
for providing training for mol% G + C studies. Dr. Sue
Eggert helped with larvae collection and identification.
Dr. James Travis and his laboratory in the Biochemistry
Department at the University of Georgia helped with the
assays of gut enzyme activity.
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Accepted April 19, 2010

C 2010 The Authors
 Insect Science (2010) 13, 303–312, DOI 10.1111/j.1744-7917.2010.01343.x

ORIGINAL ARTICLE

Mining diversity of the natural biorefinery housed within

Tipula abdominalis larvae for use in an industrial biorefinery
for production of lignocellulosic ethanol

Dana M. Cook and Joy Doran-Peterson

Microbiology Department, University of Georgia, Athens, Georgia, USA

Abstract Although they are the largest taxonomic group of animals, relatively few in-
sects have been examined for symbiotic relationships with micro-organisms. However,
this is rapidly changing because of the potential for examination of the natural insect–
microbe–lignocellulose interactions to provide insights for biofuel technology. Micro-
organisms associated with lignocellulose-consuming insects often facilitate the digestion
of the recalcitrant plant diet; therefore these microbial communities may be mined for novel
lignocellulose-degrading microbes, or for robust and inexpensive biocatalysts necessary
for economically feasible biofuel production from lignocellulose. These insect–microbe
interactions are influenced by the ecosystem and specific lignocellulose diet, and appre-
ciating the whole ecosystem–insect–microbiota–lignocellulose as a natural biorefinery
provides a rich and diverse framework from which to design novel industrial processes.
One such natural biorefinery, the Tipula abdominalis larvae in riparian ecosystems, is
reviewed herein with applications for biochemical processes and overcoming challenges
involved in conversion of lignocellulosic biomass to fuel ethanol. From the dense and
diverse T. abdominalis larval hindgut microbial community, a cellulolytic bacterial iso-
late, 27C64, demonstrated enzymatic activity toward many model plant polymers and
also produced a bacterial antibiotic. 27C64 was co-cultured with yeast in fermentation of
pine to ethanol, which allowed for a 20% reduction of commercial enzyme. In this study,
a micro-organism from a lignocellulose-consuming insect was successfully applied for
improvement of biomass-to-biofuel technology.
Key words ethanol, hydrolytic enzymes, insect-associated microorganisms, lignocellu-
lose

Introduction
Insects are the largest taxonomic group of animals on
earth. Although a few thorough studies have shown in-
sects host an environment with high microbial diversity
Correspondence: Joy Doran-Peterson, Microbiology Depart-
ment and Biofuels, Biopower, and Biomaterials Initiative, Uni-
versity of Georgia, 527 Biological Sciences Building, 1000
Cedar Street, Athens, GA 30602. Tel: 706 542 4115; fax: 706
542 2674; email: jpeterso@uga.edu
(Buchner, 1965; Tanada & Kaya, 1993; Breznak & Brune,
1994; Kane & Pierce, 1994; Moran, 2001; Dillon & Dil-
lon, 2004; Brune, 2005), less than 1% of described insect
species have been examined for micro-organisms (Kane
& Mueller, 2002). Insects that degrade lignocellulose and
host a gut microbial consortia have become of special
interest recently for the application of microbial con-
version of lignocellulose to biofuels, including ethanol,
butanol and hydrogen. One of the major challenges in
lignocellulose conversion is the need for novel, robust
and inexpensive enzymes to deconstruct lignocellulose
into fermentable sugars; the gut microbial community


C 2010 The Authors 303
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C Institute of Zoology, Chinese Academy of Sciences
304 D. M. Cook & J. Doran-Peterson
of lignocellulose-degrading insects may be mined for
these enzymes, as well as novel micro-organisms them-
selves for the application of improved biofuel production
technology.
As plants have evolved recalcitrant structures to resist
predation, so have their consumers evolved mechanisms
to overcome that resistance. For micro-organisms, these
mechanisms include lignocellulolytic enzymes that de-
construct plant polymers to sugar moieties. Some herbiv-
orous insects host a gut microbial community that facili-
tates digestion of a recalcitrant lignocellulosic diet. Often
in nature lignocellulose degradation is a cooperative ac-
tivity, which has been reported to be most effective with
a mixed culture of cellulolytic and non-cellulolytic bacte-
ria (Odom & Wall, 1983; Haruta et al., 2002; Kato et al.,
2004). These studies indicate that non-cellulolytic aerobes
enhance cellulose degradation, presumably by establish-
ing and maintaining anaerobic conditions, neutralizing
pH and consuming metabolites that might interfere with
cellulose degradation.
Individual members of microbial communities are often
most metabolically active only when in association with
other members of the community. Also, the microbial
activity, water chemistry and other biogeochemical pro-
cesses in the ecosystem, external to the insect itself, can
greatly influence functions and productivity of gut micro-
bial communities (Röling, 2007). Therefore, the study of
ecosystem–insect–microbiota–lignocellulose interactions
should be viewed as a whole process, a natural biorefin-
ery, for greater insight into the individual constituents of
microbial species and enzymes.
Macro-invertebrate shredders in riparian streams
Shredders are a functional group of macro-invertebrates
that consume lignocellulolosic detritus in small ripar-
ian stream ecosystems. In these low-order ecosystems,
leaf litter comprises the majority of carbon and en-
ergy input (Vannote et al., 1980). Thus, shredders are
an important segment of the small stream ecosystem
and usually comprise ≈ 20% of the total biomass (or
10% numerical abundance) of stream macro-invertebrates
(Petersen et al., 1989). Although leaf litter is the pri-
mary source of both carbon and energy input into small
stream systems, many organisms are unable to degrade
this lignocellulosic material, which has low nutritional
value due to a high C : N ratio. Furthermore, pro-
teins complexed with tannins, lignins and highly struc-
tured plant polysaccharide polymers (cellulose, hemicel-
lulose and lignin) make digestion of leaf litter difficult
(Martin et al., 1980). By converting lignocellulose into
a form that other organisms can use, shredders influ-
Journal compilation 
C Institute of Zoology, Chinese Academy of Sciences, Insect Science, 13, 303–312
ence the bioavailability of carbon and energy within the
ecosystem.
Shredders, and detritovores in general (in contrast to
some other insects systems such as the termite, Martin
& Martin, 1979) do not seem to produce themselves the
necessary lignocellulolytic activity to digest the abundant
plant polymers of their diets (Barlocher & Kendrick, 1974;
Barlocher & Porter, 1986; Walters & Smock, 1991). Some
shredders host gut microbial communities that are hypoth-
esized to facilitate digestion of lignocellulose (Cook et al.,
2007; Klug & Kotarski, 1980; Sinsabaugh et al., 1985).
The necessity of microbial-mediated hydrolysis and fer-
mentation for the digestion and assimilation of plant poly-
mers has been demonstrated with 14 C-labelled cellulose in
three different genera of shredders (Pteronarcys proteus,
Tipula abdominalis, and Pycnopsyche luculenta) (Sins-
abaugh et al., 1985). Acetate from microbial fermentation
was produced in the guts of shredders T. abdominalis and
Pycnopsyche guttifer and was transported across the gut
wall into the hemolymph (Lawson & Klug, 1989).
Tipula abdominalis larva: A macro-invertebrate
shredder hosting a gut microbiota
Tipula abdominalis is an aquatic crane fly in riparian
streams; the larvae are primary shredders of leaf litter
detritus. The larvae progress through four larval instar
stages. First instar larvae hatch from eggs late in sum-
mer and then progress relatively quickly (weeks) through
second and third instar stages. They molt into the fourth
instar stage in late autumn and persist longest (months) in
this final instar (Byers, 1996). Fourth instar larvae con-
sume conditioned leaf litter throughout autumn, winter
and spring. The gut morphology of T. abdominalis larvae
consists of two main compartments: midgut and hindgut.
In contrast to the linear gut morphology of other insects
(e.g., Pteronarcys spp., Pycnopsyche spp.), the anterior
portion of the hindgut of T. abdominalis protrudes from
the hindgut where material may be detained for extended
digestion; this structure has been termed a “fermentation
paunch” or “fermentation chamber” (Klug & Kotarski,
1980) (Fig. 1). The midgut is highly alkaline at pH 11,
while the hindgut is neutral at pH 7 (Martin, 1987). Studies
suggest that proteolysis occurs in the alkaline conditions
in the midgut, dissociating protein complexes from plant
polymers, which are then more accessible for sacchar-
ification and microbial fermentation in the pH-neutral
hindgut (Sinsabaugh et al., 1985; Lawson & Klug, 1989;
Garca & Barlocher, 1998; Clark, 1999; Canhoto & Garca,
2006).
Scanning electron microscopy studies revealed that
the T. abdominalis larval gut hosts a dense and diverse

C 2010 The Authors
 Natural biorefinery for industrial applications 305

Fig. 1 Drawing of T. abdominalis gut tract with viable and direct bacterial cell counts. (a) aerobic and (b) anaerobic CFU per mg dry
weight, (c) direct microscope counts per mg dry weight (Klug & Kotarski, 1980). Drawing modified from Rogers (2005).

microbial community (Klug & Kotarski, 1980; Clyde, previously described uncultured bacteria. No Clostridia
1996). The lumen contents of the midgut comprised a clones were similar to cultured bacteria at ≥ 97% se-
microbial diversity similar in morphology to that of in- quence similarity (Fig. 2A). At ≥ 97% sequence similar-
gested leaf detritus. No micro-organisms were associated ity, 92% of Bacteroidetes clones were similar to another
with the wall (larval epithial tissues) of the midgut. In con- clone, while no Bacteroidetes clones were similar to any
trast, the lumen and wall of the hindgut hosted a microbial previously described sequence (Fig. 2B). The few previ-
community of greater density and morphological diver- ously described sequences that were similar to clones in
sity, which differed from that of the ingested leaf detritus.
Aerobic and anaerobic cultivation of bacteria revealed that
colony-forming units also increased from midgut lumen to
hindgut lumen to hindgut wall. The density and diversity
of the microbial community increased with each larval
instar stage. Although a portion of the lumen contents
was maintained throughout molting, no micro-organisms
were associated with the hindgut wall immediately after
molting.
Analysis of T. abdominalis hindgut bacteria using 16S
rRNA gene libraries revealed a phylogenically diverse
community (Cook et al., 2007). From a total of 322
clones, 163 phylotypes (operational taxonomic units shar-
ing ≥ 99% sequence similarity), were identified and eval-
uated for similarity to other ribosomal RNA (rRNA) genes
from clones and isolated bacteria using database com-
parisons. Clones represented nine classes: Actinobacte-
ria, Bacteroidetes, Clostridia, Alphaproteobacteria, Be-
taproteobacteria, Gammaproteobacteria, Cyanobacteria,
Deferribacteres and Planctomycetacia. A closer look at
the clone library sequences revealed that the majority
of clones had highest sequence similarity to Clostridia
and Bacteroidetes, representing 65% and 19% of the total
clones, respectively. Clostridia and Bacteroidetes clones
were the only classes found in all four libraries. Using
methods similar to those described (Cook et al., 2007)
Clostridia and Bacteroidetes clones were compared to one
another, as well as previously described uncultured and
cultured bacteria, at varying percent sequence similarity.
Clones were more similar to one another than to previously Fig. 2 Percentage of (A) Clostridia clones, and (B) Bac-
described sequences, and more similar to uncultured than teroidetes clones similar to another clone from this study
cultured bacteria (Fig. 2). At ≥ 97% sequence similarity (squares), previously described uncultured (closed circles), or
(bacterial species level), 76% of Clostridia clones were cultured (open circles) bacteria at x% sequence similarity. Data
similar to another clone, while only 4% were similar to from Cook et al. (2007).


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C Institute of Zoology, Chinese Academy of Sciences, Insect Science, 13, 303–312
306 D. M. Cook & J. Doran-Peterson
Fig. 3 (A) Summary of isolates’ enzymatic activities on
nine model plant polymer substrates: carboxymethylcellulose
(CMC), starch, xylan, polygalacturonate (PGA), and methylum-
belliferyl conjugated to cellobiopyranoside (MUC), arabinofu-
ranoside (MUA), glucoside (MUG), mannopyranoside (MUM),
and xyloside (MUX). (B) Summary of isolates positive on 1 to
9 model substrates. Data from Cook et al. (2007).
that study were sequences from other uncultured bacte-
ria, most often cloned from other insect guts (data not
shown). These analyses of the 16S rRNA genes from the
T. abdominalis larval hindgut bacteria indicated that this
microbial community was unique from previously de-
scribed micro-organisms.
Further investigations of the T. abdominalis larval
hindgut microbiota included isolation and characteriza-
tion of bacteria (Cook et al., 2007). Using simple aero-
bic cultivation techniques, 59 isolates representing four
classes of bacteria were obtained. Those isolates were
screened for enzymatic activity on model plant polymer
substrates. Many of the isolates were able to hydrolyze
several of the substrates (Fig. 3A). A group of five iso-
lates with identical 16S rRNA gene sequences was able
to degrade all model substrates (Fig. 3B). A representa-
Journal compilation 
C Institute of Zoology, Chinese Academy of Sciences, Insect Science, 13, 303–312
tive was chosen, isolate 27C64, and putatively identified
by 16S rRNA gene sequence as Paenibacillus amolyticus.
This isolate was able to degrade all substrates tested.
Bacterial isolate 27C64
Bacterium 27C64 was isolated from the T. abdomi-
nalis larva hindgut and characterized as described (Cook
et al., 2007). Briefly, hindgut homogenates were seri-
ally diluted and plated onto tryptic soy agar and incu-
bated for up to 3 weeks at 22◦ C. Colonies were subcul-
tured until pure cultures were obtained. Sequencing of
the 16S rRNA genes from the cultured isolates was per-
formed at MIDI Labs (Newark, Delaware, USA). Isolate
27C64 had enzymatic activity (hydrolysis of substrate) on
model substrates carboxymethylcellulose (CMC) (Wood
& Kellogg, 1988); starch (Difco 272100); xylan (Mondou
et al., 1986); polygalacturonate (PGA) (Starr et al., 1977);
and methylumbelliferyl conjugated to cellobiopyranoside
(MUC), arabinofuranoside (MUA), glucoside (MUG),
mannopyranoside (MUM), and xyloside (MUX) (Shar-
rock, 1988). Further study revealed that 27C64 also pro-
duced the polymyxin antibiotics E1 and E2 (Henriksen
et al., 2007). For further characterization of 27C64, car-
bohydrate utilization was assayed and enzymatic activ-
ity was quantified. Because of its carbohydrate prefer-
ences, its possession of many plant polymer-degrading
enzymes, and its production of bacterial antibiotics,
27C64 was investigated in biomass fermentations to fuel
ethanol.
Biomass fermentations to fuel ethanol
Biochemical conversion processes such as using en-
zymes to deconstruct plant cell walls followed by fermen-
tation of the carbohydrates to generate a useful product
such as ethanol, offer great potential for expanding our
renewable chemicals and fuels capacity. Current com-
mercial ethanol production from starch involves two en-
zymes, α-amylase and glucoamylase that easily depoly-
merize starch into glucose, which is then fermented to
ethanol by Saccharomyces yeasts. Plant cell wall de-
construction from lignocellulosic biomass is more chal-
lenging due to the recalcitrance and complexity of the
biomass itself. Depending upon the biomass some type
of physical and/or chemical pretreatment is necessary to
open the fiber structures to allow enzyme access to the
plant carbohydrate polymers (Wright, 1989; Gray et al.,
2006; Farrell et al., 2006). To illustrate the use of insect-
associated micro-organisms in biofuel production, we will
discuss fermentation of pretreated pine biomass using a

C 2010 The Authors

Saccharomyces yeast strain together in co-culture with a
bacterium (27C64) isolated from the Tipula abdominalis
hindgut microbial community (Patents-Pending).
The use of enzymes to saccharify lignocellulosic
biomass, such as pine, is typically performed after other
physical and/or chemical methods of pretreatment and
can be accomplished prior to or in conjunction with
fermentation. Pretreatment breaks down biomass to al-
low access to the enzymes, which can then hydrolyze
the remaining cellulose, hemicellulose and pectin poly-
mers (Galbe & Zacchi, 2007; Himmel et al., 2007). Most
enzymatic saccharifications are performed with commer-
cially available cell-free extracts of fungal cultures, or in
some cases, bacterial cultures, designed to provide hydrol-
ysis of the lignocellulose. If the fermentation biocatalyst
and the enzyme mixtures function optimally at the same
pH and temperature range, then simultaneous sacchari-
fication and fermentation (SSF) is the desired method.
SSF is advantageous because the fermenting organism, in
this example the yeast, and the enzymes, most often com-
mercial mixtures from fungi, are added at the same time
and as the enzymes liberate sugars, the yeast converts the
sugars to ethanol, alleviating any end-product inhibition
of the enzymes due to sugar accumulation (Gauss et al.,
1976; Takagi et al., 1977; Doran-Peterson et al., 2009 and
references therein).
Degradation of cellulose is achieved through the ac-
tion of three types of enzymes: endo-glucanases, cel-
lobiohydrolases (or exo-glucanases) and β-glucosidases
(Chang, 2007; Turner et al., 2007). Endo-glucanases and
cellobiohydrolases cleave within or at the end of the glu-
can chain, respectively (the latter releasing units of cel-
lobiose), and are classified based on both their structural
fold and catalytic mechanism (Henrissat & Davies, 1997).
Table 1 Enzyme activity units (IU) per mL of culture supernatant using various substrates.
Substrate Xylanase
Glucose ND†
Mannose 0.19
Xylose 0.13
Arabinose 0.13
Cellulose 0.33
Pectin 0.14
Starch ND
Xylan 0.38
Carboxymethylcellulose 0.17
Pine acid hydrolysate‡ 0.13
†
ND = none detected.
‡
hydrolysate = the liquid fraction of the two step-sulfur dioxide, steam explosion pretreatment.

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C Institute of Zoology, Chinese Academy of Sciences, Insect Science, 13, 303–312
Natural biorefinery for industrial applications 307
β-glucosidases cleave cellobiose to monomeric glucose
and are essential for overall cellulose degradation to glu-
cose because accumulated cellobiose and/or glucose in-
hibit the activity of glucanases (Bayer et al., 1998). Addi-
tional enzyme activities useful for pretreated pine biomass
include, but are not limited to, mannanases and xylanases
(Turner et al., 2007).
To improve ethanol yields from pine biomass, 27C64
was co-cultured with yeast. It was hypothesized that en-
zymes produced by 27C64 would help to saccharify the
substrate and allow for a reduction in the fungal en-
zyme loading required to make sugars available for the
yeast conversion to ethanol. Also, antibiotic production
from 27C64 would reduce bacterial contamination of the
fermentation without impacting the performance of the
yeast.
Materials and methods
27C64 carbohydrate utilization and enzyme activity
27C64 was grown on basal minimal media with 1%
(w/v) carbon sources as listed in the Table 1. The de-
fined basal medium was based on modified Davis minimal
media containing the following ingredients per liter: 7 g
K2 HPO4 , 3 g KH2 PO4 , 1 g (NH4 )2 SO4 , 0.5 g sodium cit-
rate and 0.1 g MgSO4 -7H2 O. The original recipe includes
glucose. Glucose was not added, and the sugars listed in
Table 1 were individually added to result in 11 separate
media. After 48 h, supernatant was collected via centrifu-
gation and assayed for xylanase, polygalacturonase and
CMCase activity by measuring degradation of oat spelt
xylan, polygalacturonic acid or carboxymethylcellulose,
a modified cellulose with methylated hydroxyl groups
Polygalacturonase Carboxymethylcellulase
ND 0.12
0.25 0.46
0.23 0.09
0.23 0.15
0.18 ND
ND 0.17
0.19 ND
0.06 0.12
0.21 0.08
ND 0.19
308 D. M. Cook & J. Doran-Peterson
for increased solubility of the substrate compared to na-
tive cellulose. Degradation of carboxymethylcellulose is
suggestive of endoglucanase activity, but it is not spe-
cific for endoglucanase activity only, therefore the term
“CMCase” activity is often used (Sharrock, 1988). These
model biomass substrates were added at 1% w/v concen-
trations in 50 mmol/L sodium acetate buffer, pH 4.8 and
assayed for xylanase, polygalacturonase and CMCase ac-
tivity, respectively, using published methods (Berlin et al.,
2005; Ximenes et al., 2007). Filter paper activity (FPAase)
was assayed as described by Mandels et al. (1976).
Release of reducing sugars was determined according to
Miller (1959). Briefly, reactions were incubated at 50◦ C
for 15 min and stopped by adding 1.0 mL DNS reagent
(g/L: 10 dinitrosalicylic acid, 16 sodium hydroxide pel-
lets, 300 potassium sodium tartarate), then incubated in
a boiling water bath for 5 min. Absorbance was mea-
sured at 540 nm. Standard curves were constructed with
known glucose (CMCase and amylase), galacturonic acid
(pectinase) and xylose (xylanase) concentrations. Reduc-
ing sugars were extrapolated from standard curves to de-
termine the amount of reducing sugars liberated from the
different biomass model substrates. One unit of cellulase
(for CMC as substrate), xylanase and polygalacturonase
activity was defined as the release of one μmol of glucose,
xylose or galacturonic acid, repectively, per min.
Pine fermentation with co-culture yeast and 27C64
Pretreated G3S2 pine was produced as follows. Loblolly
pine from Georgia, USA, was chipped to a particle size of
10 mm or less. Chips were then pretreated with gaseous
sulfur dioxide in two steps. A batch of a known amount of
chips was treated with 2.5% SO2 w/w of moisture content
in chips, at a temperature of 190◦ C for 5 min. Following
this pretreatment step, the material was pressed using a
hydraulic press to collect liquid. This liquid was not used
in the experiments described herein. The pretreated solids
(material remaining after the liquid was pressed out and
removed), was then washed with water and pressed to a
dry matter content of 40%. A second impregnation with
2.5% SO2 w/w of moisture content in the solids followed,
and the materials were allowed to react at a temperature
of 210◦ C for 5 min. The sample obtained using these two
steps of pretreatment were used in co-culture fermenta-
tions. Moisture content of the pretreated pine was 71.53%.
Fermentation with yeast with and without addition of
27C64 co-culture: four bioreactors each containing 20 g
dry weight (DW; 10% solids) of pretreated pine were auto-
claved at 121◦ C. Enzymes were added on a unit per gram
dry weight of pretreated pine basis. Novozyme cellulase
cocktail (12 FPU/g or 15 FPU/g as indicated) and Cel-
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C Institute of Zoology, Chinese Academy of Sciences, Insect Science, 13, 303–312
lobiase (60 U/g) (Novozymes, Inc. Franklinton, NC, US)
were used. Active dried yeast (ADY, North American Bio-
products Corporation, Duluth, GA, US) was inoculated at
a concentration of 2 g/L in each vessel. Either sterile wa-
ter or resuspended bacterial pellets of 27C64 were added
to the bioreactor to determine the effect of co-culture on
ethanol yield. Five hundred milliliters of overnight-grown
culture of 27C64 was centrifuged, the pellet resuspended
in a small volume of 2 × tryptic soy broth and 5 × 107
cells (roughly 0.2 g DW of bacteria) were added to the
bioreactor. The total volume of each fermentation was
200 mL. Fermentation reaction was incubated at 37◦ C for
48 h with constant stirring.
To quantify ethanol production, gas chromatography
(GC) was performed (Shimadzu GC-8A; Shimadzu Corp.,
Kyoto, Japan) with column DB-624 (Agilent Technolo-
gies, Inc., Santa Clara, CA, US) as described previously
(Doran-Peterson et al., 2009). The grams of ethanol pro-
duced per FPU of cellulase calculated were calculated
as follows: g ethanol/total FPU; g ethanol = ethanol
(g/L) × 0.2 L (reaction volume); total FPU = g solids
(pine substrate) × FPU/g (12 or 15).
Results and discussion
27C64 carbohydrate utilization and enzyme activity
Cellulase enzyme activity measured as filter paper units
of activity (FPU) using xylose, mannose, or both sugars
together as growth substrates in minimal media, resulted
in 0.26, 0.20 and 0.24 FPU/mL of culture supernatant,
respectively. Strain 27C64 produces xylanase, pectinase
and cellulase when grown in the minimal basal media in
the presence of various carbon sources (Table 1).
Pine fermentation with co-culture yeast and 27C64
Ethanol production from pretreated pine at 10% w/v
solids with and without co-inoculation with the bacterium
27C64 is presented in Table 2. The theoretical maximum
for ethanol production from pretreated pine under these
conditions was 31.8 g ethanol per liter of fermentation
broth. All bioreactor-run data are the average of duplicates
due to limited pretreated pine availability. Yeast cells were
evaluated for their ability to produce ethanol without the
addition of any enzymes (neither fungal nor from 27C64)
as a baseline. Yeast cells plus an inoculum of 27C64 cells
only (without the additional fungal enzymes and without
the 27C64 culture supernatant) were cultivated together
to determine whether 27C64 could grow in the pine sub-
strate without decreasing the ethanol produced by the
fermenting yeasts. Where fungal enzymes were added,

C 2010 The Authors

Table 2 Ethanol production in g/L after 24 and 48 h of fermentation using commercially available fungal enzymes and either yeast
cells alone or in combination with 27C64 cells.
Fungal
cellulase FPU/g†
g/L ethanol Time (h) 0
24
48
g ethanol/FPU fungal cellulase
Each value represents the average of duplicates for G3S2 pretreated pine fermentations. † FPU = filter paper units of activity per gram
dry weight of pretreated pine.
fermentations contained 60 U fungal cellobiase per gram
dry weight (gdw) of pretreated pine solids. Four bioreac-
tors contained 12 FPU cellulase/gdw pretreated pine in
addition to the cellobiase and two of the bioreactors con-
tained 15 FPU cellulase/gdw of pretreated pine in addition
to the cellobiase. Two of the 12 FPU/gdw pretreated pine
bioreactors were inoculated with 27C64 cells at the same
time as yeast and commercial enzyme addition. Two addi-
tional bioreactors were inoculated with yeast and 27C64
cells at the same time without any additional fungal com-
mercial enzymes.
A small but significant amount of ethanol was produced
during the 48 h of fermentation with the 27C64 inoculum
compared to the yeast inoculum alone. This suggests that
27C64 was able to produce enzymes able, at least to some
extent, to degrade the pretreated pine substrate. In ad-
dition, approximately 17% more ethanol was produced
from the fermentation where 27C64 was added together
with the yeast during fermentations using 12 FPU cellu-
lose/gdw pretreated pine. In contrast, increasing the fungal
enzyme loading from 12 FPU to 15 FPU/gdw pretreated
pine increased the ethanol concentration maximum by
only 5% (1.2 g/L). The 17% increase in ethanol produc-
tion in the fermentations with added 27C64 may not be a
major increase; however, it does show that adding 27C64
cells at the start of the fermentation does not negatively
impact ethanol production from the fermenting yeast. This
is a bit surprising because 27C64 is capable of metabo-
lizing some of the same sugars as the yeast. However, the
27C64 strain can use sugars that the yeast is unable to me-
tabolize, such as pentoses. Ongoing studies with 27C64
(data not shown) suggest that this organism can use acetate
and other potentially inhibitory compounds as a carbon
source, perhaps helping to detoxify the yeast’s environ-
ment; however, more studies are needed to confirm this
hypothesis.
Although these conditions have not been opti-
mized for the mixture of 27C64 bacteria, fungal

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Journal compilation 
C Institute of Zoology, Chinese Academy of Sciences, Insect Science, 13, 303–312
Natural biorefinery for industrial applications 309
Yeast cells only Yeast and 27C64
0 12 15 0 12
0.6 0.55 0.7 0.7 0.7
0.9 17.8 22.9 3.0 23.5
1.3 25.4 26.6 6.2 29.8
NA 0.021 0.018 NA 0.025
enzymes and fermenting yeasts, the authors believe
the results support the proof in principle of using
insect-associated micro-organisms for enhancing biofuel
production.
Tipula abdominalis larva: a natural biorefinery
The model of the T. abdominalis larva as a natural biore-
finery can be applied toward developments in technology
for industrial biomass refinery processes (Fig. 4). In this
model, the substrate (conditioned leaf litter) is ingested by
larvae. Maceration of the substrate during ingestion de-
creases particle size and increases surface area-to-volume
ratios. Upon entering the alkaline midgut, proteolysis de-
grades complexed proteins making polysaccharide poly-
mers more accessible for further processing. In the neu-
tral pH hindgut, bacterial enzymes saccharify cellulose
and hemicellulose. These sugars are then consumed by
bacteria and converted to acetate and other fermenta-
tion products, which can be transported across the gut
to the hemolymph to support larval energy and growth re-
quirements (Lawson & Klug, 1989). In the fermentation
paunch, material may be retained for extended processing
(Klug & Kotarski, 1980). Lastly, waste and by-products
are excreted and are valuable to other organisms in the
ecosystem.
The simplicity of this model should not overshadow
the true complexity of this system. In this natural biore-
finery, numerous bacterial species are interacting in com-
plex relationships to degrade and ferment a heterogeneous
substrate in a simultaneous saccharification and fermen-
tation (SSF) approach. In another possible approach em-
ployed by industrial biomass refineries, a separate hy-
drolysis followed by fermentation (SHF) approach, in
which biomass is converted in discrete and separate steps:
enzymatic saccharification followed by fermentation. In
a third process strategy, partial saccharification and co-
fermentation (PSCF), enzyme saccharification is begun
310 D. M. Cook & J. Doran-Peterson
Fig. 4 Model of the T. abdominalis larva as a natural biorefinery.
prior to fermentation, then allowed to proceed (though at
decreased efficiency) during the fermentation process. In
contrast to the natural biorefinery of the T. abdominalis
hindgut, industrial biomass refineries typically require
fungal enzymes for saccharification and a single micro-
bial species in monoculture is employed for fermentation
of a relatively homogeneous substrate. This can present
problems for SSF processes, as fungal enzymes and fer-
menting micro-organisms often have different optimal
conditions. Study of the natural biorefinery can provide
insights to microbe interactions during lignocellulose con-
version, including cooperative cellulose degradation and
how to deal with inhibitors produced during lignocellu-
lose deconstruction. Like the natural biorefinery, indus-
trial biomass refineries should seek value in processing
by-products that may otherwise be considered waste. This
natural biorefinery is also a reservoir for potentially novel
enzymes that can enhance biomass degradation processes.
Another application is to genetically engineer the genes
for these enzymes into designer fermenting organisms ca-
pable of selected pre-treatment of the biomass to be con-
verted. Novel enzymes, and possibly even novel metabolic
pathways, can be genetically engineered into ferment-
ing micro-organisms to increase their value as industrial
biocatalysts.
Acknowledgments
This work was funded in part by the University of
Georgia Microbiology Department, C2 Biofuels (Atlanta,
GA), and an award from the Technology Commercial-
ization Office of the University of Georgia Research
Foundation, Inc. The authors wish to thank anonymous
reviewers for their contributions toward improving the
manuscript.
Journal compilation 
C Institute of Zoology, Chinese Academy of Sciences, Insect Science, 13, 303–312
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Accepted April 12, 2010

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